Methods and compositions for treatment of glycogen storage diseases and glycogen metabolism disorders

ABSTRACT

The present disclosure provides for compositions comprising a chimeric polypeptide comprising a polypeptide effective for treating glycogen storage disease and an internalizing moiety that promotes delivery into cells. In certain embodiments, the polypeptide effective for treating glycogen storage disease is an acid alpha-glucosidase (GAA), a laforin, an amyloglucosidase (AGL), a malin, or an alpha amylase. The present disclosure also provides for methods for decreasing glycogen accumulation in cells or for treating glycogen storage diseases, including Forbes-Cori Disease, Andersen Disease, von Gierke Disease, Pompe Disease, and Lafora Disease, comprising administering the chimeric polypeptide disclosed herein.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/318,256, filed Dec. 12, 2016, which is a national stage filing under35 U.S.C. 371 of International Application No. PCT/US2015/035680, filedJun. 12, 2015, which claims the benefit of priority to U.S. provisionalapplication Ser. No. 62/012,151, filed Jun. 13, 2014, U.S. provisionalapplication Ser. No. 62/042,755, filed Aug. 27, 2014, U.S. provisionalapplication Ser. No. 62/042,689, filed Aug. 27, 2014, and U.S.provisional application Ser. No. 62/096,735, filed Dec. 24, 2014. Theentire contents of each of the foregoing applications are herebyincorporated by reference in their entirety. International ApplicationNo. PCT/US2015/035680 was published under PCT Article 21(2) in English.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via and is hereby incorporated by reference in its entirety.Said ASCII copy, created on Feb. 11, 2019, is named Seq_Lst.txt, and is12,288 bytes in size.

BACKGROUND OF THE DISCLOSURE

Glycogen storage diseases and glycogen metabolism disorders are a seriesof diseases that are caused by defects in basic metabolizing enzymes,thereby resulting in defects in glycogen synthesis or breakdown withinmuscles, liver, neurons and other cell types. Glycogen storage diseasesmay be either genetic (usually as autosomal recessive disorders) oracquired (e.g., by intoxication with alkaloids) (Monga et al., 2011,Molecular Pathology of Liver Diseases, Molecular Pathology Library 5,Chapter 45). There are a number of different types of glycogen storagediseases, including GSDs Types I-XI, GSD Type 0, as well as Laforadisease which is often termed a glycogen metabolism disorder. Thesediseases differ with regard to the enzyme that is mutated and/or primarytissue affected (Monga et al. and Gentry, et al., 2013, FEBS J,280(2):525-37).

a. Forbes-Cori Disease

Forbes-Cori Disease, also known as GSD Type III, GSD III, or glycogendebrancher deficiency, is an autosomal recessive neuromuscular/hepaticdisease with an estimated incidence of 1 in 100,000 births. These termsare used interchangeably throughout. Forbes-Cori Disease representsapproximately 27% of all Glycogen Storage Disorders. The clinicalpicture in Forbes-Cori Disease is reasonably well established butexceptionally variable. Although generally considered a disease of theliver, with hepatomegaly and cirrhosis, Forbes-Cori Disease also ischaracterized by abnormalities in a variety of other systems. Muscleweakness, muscle wasting, hypoglycemia, dyslipidemia, and occasionallymental retardation also may be observed in this disease. Some patientspossess facial abnormalities. Some patients also may be at an increasedrisk of osteoporosis. Different patients may suffer from one, or morethan one, of these symptoms. The differences in clinical manifestationsof this disease are often associated with different subtypes of thisdisease.

There are four subtypes of Forbes-Cori Disease. The Type A subtypeaccounts for approximately 80% of the cases, lacks enzymatic activity(e.g., both glucosidase and transferase activities associated withnative enzymatic activity) and affects both the liver and muscle. TheType B subtype accounts for approximately 15% of the cases, lacksenzymatic activity (e.g., both glucosidase and transferase activitiesassociated with native enzymatic activity) and affects only the liver.The Type C and D subtypes account for less than 5% of the cases, areassociated with selective loss of glucosidase activity (Type C) ortransferase activity (Type D) and are clinically similar to the Type Asubtype.

Forbes-Cori Disease is caused by mutations in the AGL gene. The AGL geneencodes the amylo-1,6-glucosidase (AGL) protein (GenBank Accession Nos.NP_000019.2; NM_000645.2; and NM_000646.2) which is a cytoplasmic enzymeresponsible for catalyzing the cleavage of terminal α-1,6-glucosidelinkages in glycogen and similar molecules. The AGL protein has twoseparate enzymatic activities: 4-alpha-glucotransferase activity andamylo-1,6-glucosidase activity. Both catalytic activities are requiredfor normal glycogen debranching activity. Glycogen is a highly branchedpolymer of glucose residues.

AGL is responsible for transferring three glucose subunits of glycogenfrom one parallel chain to another, thereby shortening one linear branchwhile lengthening another. Afterwards, the donator branch will stillcontain a single glucose residue with an alpha-1,6 linkage. Thealpha-1,6 glucosidase of AGL will then remove that remaining residue,generating a “de-branched” form of that chain on the glycogen molecule.Without proper glycogen de-branching, as occurs in the absence offunctional AGL, abnormal glycogens resembling an amylopectin-likestructure (polyglucosan) result and accumulate in various tissues in thebody, including hepatocytes and myocytes. This abnormal form of glycogenis typically insoluble and may be toxic to cells.

Currently, the primary treatment for Forbes-Cori is dietary and is aimedat maintaining normoglycemia (Ozen, et al., 2007, World J Gastroenterol,13(18): 2545-46). To achieve this, patients are fed frequent meals highin carbohydrates and cornstarch supplements. Patients having myopathyare also fed a high-protein diet. Liver transplantation resolves allliver-related biochemical abnormalities, but the long-term effect ofliver transplantation on myopathy/cardiomyopathy is unknown (Ozen etal., 2007). These tools for managing Forbes-Cori are inadequate. Dietaryregimens have significant compliance problems—particularly with youngpatients. As such, there is a need for a Forbes-Cori therapy thatreduces the build-up of glycogen and/or polyglucosan .

b. Andersen's Disease

Glycogen storage disease type IV (GSD IV), also known as AndersenDisease, Andersen's Disease or amylopectinosis (and these terms are usedinterchangeably herein), is a rare autosomal recessive disorder causedby deficiency of the glycogen branching enzyme (GBE) (GenBank AccessionNo. NP_000149.3), also called amylo-(1,4 to 1,6) transglycosylase. GBEproduces a-1,6 branches in glucose through a process involving theterminal transfer of a terminal fragment of 6-7 glucose residues (from apolymer of at least 11 glucose residues in length) to an internalglucose residue at the C-6 hydroxyl position. In humans, the GBE1 geneis present on chromosome 3p12 and encodes a peptide having 702 aminoacids.

Reduced or absent levels of GBE result in tissue accumulation ofabnormal glycogen with fewer branch points and longer outer branchesthat resembles an amylopectin-like structure, also known as polyglucosan(Lee, et al., 2010, Hum Mol Genet, 20(3):455-465). Polyglucosan has lowsolubility and may form precipitates in the liver, heart and muscle.

Andersen disease is clinically heterogeneous, with variable tissueinvolvement and age of onset (Akman, 2014, Neurology, 82(1):P1.054). Theage of onset ranges from fetus to adulthood and is divided into fourgroups: (i) perinatal, presenting as fetal akinesia deformation sequenceand perinatal death; (ii) congenital (infantile), with hydrops fetalis,neuronal involvement and death in early infancy; (iii) childhood(juvenile), with myopathy or cardiomyopathy; and (iv) adult, withisolated myopathy or adult polyglucosan body disease (Lee, et al.,2010). Absence of enzyme activity is usually lethal in utero or ininfancy, affecting primarily muscle and liver. However, residual enzymeactivity (5-20%) leads to a juvenile or adult-onset disorder thataffects primarily muscle and both central and peripheral nervoussystems. Patients having juvenile Andersen Disease, which is the mostcommon form of Andersen Disease, first display symptoms within the firstfew months of life and are characterized by hepatosplenomegaly andfailure to thrive. The juvenile cases then typically progress to livercirrhosis, portal vein hypertension, esophageal varices and ascites,with death usually occurring by five years of age. Adult cases ofAndersen Disease may manifest similar symptoms as juvenile cases, butthe onset of these symptoms does not occur until later in the patient'slifetime.

Treatment of Andersen Disease is usually dietary, by maintaining bloodglucose along with adequate nutrient intake in order to improve liverfunction and muscle strength. In cases of progressive liver failure,liver transplants may be employed. Similar to the therapies forForbes-Cori Disease, these tools for managing Andersen Disease areinadequate and the disease is or can be fatal. As such, there is a needfor an Andersen Disease therapy that reduces glycogen and/orpolyglucosan accumulation.

c. von Gierke's Disease

Glycogen storage disease type I (GSD I) or von Gierke's disease (alsoreferred to in the art and herein interchangeably as von GierkeDisease), is the most common of the glycogen storage diseases with anincidence of approximately 1 in 50,000 to 100,000 births. The deficiencyimpairs the ability of the liver to produce free glucose from glycogenand from gluconeogenesis. Since these are the two principal metabolicmechanisms by which the liver supplies glucose to the rest of the bodyduring periods of fasting, it causes severe hypoglycemia and results inincreased glycogen storage in liver and kidneys. This can lead toenlargement of both organs.

The most common forms of GSD I are designated GSD1a and GSD1b, theformer accounting for over 80% of diagnosed cases and the latter forless than 20%. A few rarer forms have been described. GSD1a results frommutations of G6PC, the gene for glucose-6-phosphatase. GSD1b resultsfrom mutations of the SLC37A4, the glucose-6-phosphatase transporter.

Clinical manifestations result, directly or indirectly, from: theinability to maintain an adequate blood glucose level during thepost-absorptive hours of each day; organ changes due to glycogenaccumulation; excessive lactic acid generation; and damage to tissuefrom hyperuricemia. Glycogen accumulation includes accumulation in theliver and in the kidneys and small intestines. Hepatomegaly, usuallywithout splenomegaly, begins to develop in fetal life and is usuallynoticeable in the first few months of life. By the time the child isstanding and walking, the hepatomegaly may be severe enough to cause theabdomen to protrude.

The kidneys are usually 10 to 20% enlarged with stored glycogen. Thisdoes not usually cause clinical problems in childhood, with theoccasional exception of a Fanconi syndrome with multiple derangements ofrenal tubular reabsorption, including proximal renal tubular acidosiswith bicarbonate and phosphate wasting. However, prolonged hyperuricemiacan cause uric acid nephropathy. In adults with GSD I, chronicglomerular damage similar to diabetic nephropathy may lead to renalfailure.

Hepatic complications have been serious in some patients. Adenomas ofthe liver can develop in the second decade or later, with a small chanceof later malignant transformation to hepatoma or hepatic carcinomas.Additional problems reported in adolescents and adults with GSD I haveincluded hyperuricemic gout, pancreatitis, and chronic renal failure.

Treatment of von Gierke's disease is usually dietary, by frequentfeedings of foods high in glucose or starch (which is readily digestedto glucose), with the primary treatment goal being prevention ofhypoglycemia and the secondary metabolic derangements. Particularly inchildren, this requires feedings throughout the night. Two methods havebeen used to achieve this goal in young children: (1) continuousnocturnal gastric infusion of glucose or starch; and (2) night-timefeedings of uncooked cornstarch. However, there remains a need for vonGierke's disease therapies, for example, therapy that reduces glycogenaccumulation in the liver and/or kidney of patients with GSD I, such asGSD Ia or GSD Ib.

d. Lafora Disease

Lafora Disease, also called Lafora progressive myoclonic epilepsy orMELF, is a rare, fatal neurodegenerative disorder characterized by theaccumulation of insoluble, poorly branched, hyperphosphorylated glycogenin cells from most tissues of affected individuals, including the brain,heart, liver, muscle and skin. Lafora Disease patients typically firstdevelop symptoms in adolescence. Symptoms include temporary blindness,depression, seizures, drop attacks, myoclonus, ataxia, visualhallucinations, absences, and quickly developing and severe dementia.Death usually occurs 2-10 years (5 years mean) after onset.

The prevalence of Lafora Disease is unknown. While this disease occursworldwide, it is most common in Mediterranean countries, parts ofCentral Asia, India, Pakistan, North Africa and the Middle East. InWestern countries, the prevalence is estimated to be below 1/1,000,000.

Lafora Disease is an autosomal recessive disorder caused by mutations inone of two genes: EPM2A and EPM2B. EPM2A encodes for the 331 amino acidprotein known as laforin, which comprises an amino-terminal carbohydratebinding module and a carboxy-terminal dual specificity phosphatasedomain. EPM2B encodes for the E3 ubiquitin ligase known as malin.Together, laforin and malin make up a functional complex which isbelieved to be involved in negatively regulating glucose uptake bymodulating the subcellular localization of glucose transporters. Singhet al., 2012, Mol Cell Biol, 32(3):652-663. Recent studies also suggestthat the accumulation of glycogen is responsible for neurodegenerationand impaired autophagy observed in the brains of Lafora patients. Duranet al., 2014, Hum Mol Genet, 23(12): 3147-56.

There is currently no cure or effective treatment for patients havingLafora Disease. However, the seizures and myoclonus can be managed, atleast in early stages of the disease, with antiepileptic medications.

SUMMARY OF THE DISCLOSURE

There is a need in the art for methods and compositions for clearingglycogen build-up, particularly cytoplasmic glycogen build-up, or fortreating the cytotoxic effects associated with glycogen build-up, inpatients with Forbes-Cori and/or Andersen Disease and/or von GierkeDisease and/or Pompe Disease and/or Lafora Disease, as well as a needfor alternative therapies for treating any one or more of thesediseases. The present disclosure provides such methods and compositions.For example, there exists a need for decreasing glycogen accumulationin, for example, cytoplasm of cells, such as muscle and/or liver and/orkidney and/or neuronal cells. By way of further example, such methodsand compositions may decrease glycogen accumulation in lysosomes and/orthe nucleus, as an alternative to or in addition to decreasingcytoplasmic glycogen accumulation. In certain embodiments, the methodsand compositions are useful for decreasing glycogen accumulation incytoplasm, as well as in the lysosome (and, optionally, in the nucleusfor conditions characterized by nuclear accumulation of glycogen). Inthe context of these conditions, decreasing cytoplasmic glycogenbuild-up refers to decreasing accumulation of normal and/or abnormalglycogen, and may similarly apply to decreasing glycogen accumulation inother sites. Accordingly, throughout the application, references toclearing glycogen build-up or decreasing glycogen accumulation (or liketerms) encompass, unless otherwise specified, clearing or decreasingexcess (e.g., beyond normal physiological level) glycogen, includingclearing or decreasing excess glycogen present in an abnormal form(e.g., polyglucosan). In certain embodiments, the disclosure providesmethods of clearing or decreasing excess polyglucosan (e.g., clearing ordecreasing polyglucosan accumulation), such as in cytoplasm, such as inone or more of muscle cells (skeletal and/or cardiac), liver, kidney, orneurons. In certain embodiments, clearing glycogen build-up ordecreasing glycogen accumulation (or like terms) refers to doing so in,at least, cytoplasm of one or more affected cells. In certainembodiments, clearing glycogen build-up or decreasing glycogenaccumulation, such as in, at least, cytoplasm, is or comprises clearingpolyglucosan build-up or decreasing polyglucosan accumulation, such asin, at least, cytoplasm. Such methods and compositions would improvetreatment of Forbes-Cori and/or Andersen Disease and/or von GierkeDisease and/or Pompe Disease and/or Lafora Disease, particularly inpatients whose disease is severe enough and/or advanced enough to havesignificant abnormal cytoplasmic glycogen accumulation (e.g., of normaland/or abnormal glycogen). The present disclosure provides such methodsand compositions. In certain embodiments, the methods and compositionsprovided herein decrease glycogen build-up (e.g., such as clear glycogenbuild-up or decrease glycogen accumulation) in, at least, the cytoplasm.In certain embodiments, the methods and compositions of the presentdisclosure decrease polyglucosan build-up (e.g., build-up in, at least,the cytoplasm of cell(s), such as muscle and/or liver and/or neuronaland/or glial cell(s)). In certain embodiments, the methods andcompositions of the present disclosure decrease glycogen, such aspolyglucosan, build-up in, at least, cytoplasm of, at least muscleand/or liver.

One benefit of the methods and compositions provided herein is that asingle protein (e.g., a chimeric protein comprising a GAA polypeptideportion, as described herein, and an internalizing moiety portion, asdescribed herein) can be used in the study or treatment of multipleglycogen storage disorders—specifically in Forbes-Cori Disease, AndersenDisease and Pompe Disease. In certain embodiments, the chimeric proteinmay be used to treat von Gierke Disease or to promote deliver into cellsindicative of von Gierke Disease. In certain embodiments, the chimericprotein may be used to treat Lafora Disease or to promote deliver intocells indicative of Lafora Disease. Accordingly, in certain embodiments,the present disclosure provides such methods and compositions suitablefor treating any one of, any two of, any three of, any four of, or allfive of Pompe Disease, Forbes-Cori Disease, Andersen Disease, von GierkeDisease and Lafora Disease. In addition to methods and compositionsprovided herein based on protein therapeutics comprising a GAA portionand an internalizing moiety portion, the disclosure also providesmethods and compositions in which a single chimeric protein (e.g., aprotein therapeutic comprising a non-internalizing moiety polypeptideportion selected from a GAA, laforin, alpha-amylase, malin and/or AGLpolypeptide portion, as described herein, and an internalizing moietyportion, as described herein) can be used in the treatment or study ofmultiple glycogen storage disorders—specifically in the treatment orstudy of any one, any two, any three, any four or any five of theforegoing diseases. In certain embodiments, such a chimeric proteincomprising a laforin polypeptide portion is used in the treatment orstudy of Lafora Disease. In certain embodiments, such a chimeric proteincomprising a malin polypeptide portion is used in the treatment or studyof Lafora Disease. In certain embodiments, such a chimeric proteincomprising an AGL polypeptide portion is used in the treatment or studyof Lafora disease. In certain embodiments, such a chimeric proteincomprising an alpha-amylase polypeptide portion is used in the treatmentor study of Lafora Disease. In certain embodiments, such a chimericprotein comprising an alpha-amylase polypeptide portion is used in thetreatment or study of Forbes-Cori Disease. Similarly any of theforegoing chimeric polypeptides of the disclosure are useful fordecreasing glycogen accumulation in cells, in vitro and/or in vivo,including in cells from a subject or animal model of any of thesediseases. All such in vitro and in vivo methods are expresslycontemplated.

In certain embodiments, chimeric polypeptides comprising any of the GAApolypeptides disclosed herein and any of the internalizing moietiesdescribed herein can be used to treat any one or more of Pompe Disease,Forbes-Cori Disease, Andersen Disease, von Gierke Disease or LaforaDisease or can be used to decrease glycogen accumulation in cells, suchas cells of subjects having any of the foregoing diseases. In certainembodiments, such methods are in vivo methods. In certain embodiments,chimeric polypeptides comprising any of the AGL polypeptides disclosedherein and any of the internalizing moieties described herein can beused to treat Lafora Disease or can be used to decrease glycogenaccumulation in cells, such as cells of subjects having Lafora Disease.In certain embodiments, chimeric polypeptides comprising any of themalin polypeptides disclosed herein and any of the internalizingmoieties described herein can be used to treat Lafora Disease or can beused to decrease glycogen accumulation in cells, such as cells ofsubjects having Lafora Disease. In certain embodiments, chimericpolypeptides comprising any of the alpha-amylase polypeptides disclosedherein and any of the internalizing moieties described herein can beused to treat Lafora Disease or can be used to decrease glycogenaccumulation in cells, such as cells of subjects having Lafora Disease.In certain embodiments, chimeric polypeptides comprising any of thealpha-amylase polypeptides disclosed herein and any of the internalizingmoieties described herein can be used to treat Forbes-Cori Disease orcan be used to decrease glycogen accumulation in cells, such as cells ofsubjects having Forbes-Cori Disease. In certain embodiments, chimericpolypeptides comprising any of the laforin polypeptides disclosed hereinand any of the internalizing moieties described herein can be used totreat Lafora Disease or can be used to decrease glycogen accumulation incells, such as cells of subjects having Lafora Disease. In certainembodiments, a subject or cells may be treated with one or moredifferent types of any of the chimeric polypeptides disclosed herein.For example, in some embodiments, a subject may be treated with anycombination of: a chimeric polypeptide comprising any of the GAApolypeptides disclosed herein, a chimeric polypeptide comprising any ofthe laforin polypeptides disclosed herein, a chimeric polypeptidecomprising any of the AGL polypeptides disclosed herein, a chimericpolypeptide comprising any of the alpha-amylase polypeptides disclosedherein, or a chimeric polypeptide comprising any of the malinpolypeptides disclosed herein. In particular embodiments, a LaforaDisease subject is treated, in certain embodiments, with at least twochimeric polypeptides selected from the group consisting of: a chimericpolypeptide comprising any of the laforin polypeptides disclosed herein,a chimeric polypeptide comprising any of the AGL polypeptides disclosedherein, a chimeric polypeptide comprising any of the alpha-amylasepolypeptides disclosed herein, and a chimeric polypeptide comprising anyof the malin polypeptides disclosed herein.

In certain embodiments, the disclosure provides for a method of treatingAndersen Disease in a subject in need thereof. In certain embodiments,the method comprises administering a chimeric polypeptide comprising:(i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAA polypeptidecomprising or consisting of mature GAA) and (ii) an internalizingmoiety. In certain embodiments, the internalizing moiety promotesdelivery into cells. The two portions of the chimeric polypeptide may beassociated via any of a number of mechanisms (e.g., interconnected viaone or more connections, such as one or more of chemical conjugation, asa part of a fusion protein, disulfide bonds, etc). In certainembodiments, the disclosure provides for a method of decreasing glycogenaccumulation in cytoplasm of cells, such as cells of a subject havingAndersen Disease, comprising contacting such cells with (such as byadministering) a chimeric polypeptide, which chimeric polypeptidecomprises (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising or consisting of mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cells. In certain embodiments, the subject has aperinatal form of Andersen Disease. In certain embodiments, the subjecthas a congenital form of Andersen Disease. In certain embodiments, thesubject has a juvenile form of Andersen Disease. In certain embodiments,the subject has an adult form of Andersen Disease.

In certain embodiments, the disclosure provides for a method of treatingForbes-Cori Disease in a subject in need thereof. In certainembodiments, the method comprises administering a chimeric polypeptidecomprising: (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising or consisting of mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotesdelivery into cells. In certain embodiments, the disclosure provides fora method of decreasing glycogen accumulation in cytoplasm of cells, suchas cells of a subject having Forbes-Cori Disease. In certainembodiments, the method comprises contacting muscle cells with oradministering a chimeric polypeptide, which chimeric polypeptidecomprises (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising or consisting of mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cytoplasm of cells.

In certain embodiments, the disclosure provides for a method of treatingvon Gierke Disease in a subject in need thereof, comprisingadministering a chimeric polypeptide comprising: (i) an acidalpha-glucosidase (GAA) polypeptide (e.g., a GAA polypeptide comprisingor consisting of mature GAA) and (ii) an internalizing moiety, such asan internalizing moiety that promotes delivery into cells.

In certain embodiments, the disclosure provides for a method ofdecreasing glycogen accumulation in cytoplasm of cells, such as cells ofa subject having von Gierke Disease. In certain embodiments, the methodcomprises contacting liver cells with a chimeric polypeptide, whichchimeric polypeptide comprises (i) an acid alpha-glucosidase (GAA)polypeptide (e.g., a GAA polypeptide comprising or consisting of matureGAA) and (ii) an internalizing moiety, such as an internalizing moietythat promotes transport into cytoplasm of cells. In certain embodiments,the subject or the cells has a mutation in the gene encodingglucose-6-phosphatase. In certain embodiments, the subject has amutation in the gene encoding SLC37A4.

In certain embodiments, the disclosure provides for a method of treatingLafora Disease in a subject in need thereof, comprising administering achimeric polypeptide comprising: (i) an acid alpha-glucosidase (GAA)polypeptide (e.g., a GAA polypeptide comprising or consisting of matureGAA) and (ii) an internalizing moiety, such as an internalizing moietythat promotes delivery into cells. In certain embodiments, thedisclosure provides for a method of decreasing glycogen accumulation incytoplasm of cells, such as cells of a subject having Lafora Disease. Incertain embodiments, the method comprisescontacting neuronal cells witha chimeric polypeptide, which chimeric polypeptide comprises (i) an acidalpha-glucosidase (GAA) polypeptide (e.g., a GAA polypeptide comprisingor consisting of mature GAA) and (ii) an internalizing moiety thatpromotes transport into cytoplasm of cells. In certain embodiments, thesubject or cells has a mutation in the EPM2A gene. In certainembodiments, the subject or cells has a mutation in the EPM2B gene.

In certain embodiments of any of the methods of treatment disclosedherein, the subject in need thereof is a subject having pathologiccytoplasmic glycogen accumulation prior to initiation of treatment withsaid chimeric polypeptide.

In certain embodiments of any of the methods described herein, themethod is an in vitro method and cells are contacted in vitro. Incertain embodiments of any of the methods described herein, the methodis an in vivo method and cells are contacted in vivo, such as byadministering to a subject.

In certain embodiments, the chimeric polypeptide for use in any of themethods disclosed herein comprises any of the GAA polypeptides describedherein. In certain embodiments, the chimeric polypeptide for use in anyof the methods disclosed herein comprises the GAA polypeptide set forthin SEQ ID NO: 1 or 2. In certain embodiments, the chimeric polypeptidedoes not comprise the portion of GAA polypeptide set forth in residues1-56 of SEQ ID NO: 1 or 2. In certain embodiments, the chimericpolypeptide does not comprise the portion of GAA polypeptide set forthin residues 1-57 of SEQ ID NO: 1 or 2. In certain embodiments, thechimeric polypeptide lacks at least a portion of the GAA full linkerregion, wherein the full linker region corresponds to the amino acids57-78 of SEQ ID NOs: 1 or 2. In certain embodiments, the chimericpolypeptide lacks at least a portion of the GAA full linker region,wherein the full linker region corresponds to the amino acids 57-78 ofSEQ ID NOs: 1 or 2. In certain embodiments, neither the GAA polypeptidenor the chimeric polypeptide comprise a contiguous amino acid sequencecorresponding to the amino acids 1-60 of SEQ ID NO: 1 or 2. In certainembodiments, the chimeric polypeptide or GAA polypeptide comprises theamino acid sequence of SEQ ID NO: 21. In certain embodiments, neitherthe GAA polypeptide nor the chimeric polypeptide comprise a contiguousamino acid sequence corresponding to the amino acids 1-66 of SEQ ID NO:1 or 2. In certain embodiments, the chimeric polypeptide or GAApolypeptide comprises the amino acid sequence of SEQ ID NO: 22. Incertain embodiments, neither the GAA polypeptide nor the chimericpolypeptide comprise a contiguous amino acid sequence corresponding tothe amino acids 1-69 of SEQ ID NO: 1 or 2. In certain embodiments, thechimeric polypeptide or GAA polypeptide comprises the sequence of SEQ IDNO: 23. In certain embodiments, the mature GAA polypeptide has amolecular weight of approximately 70-76 kilodaltons. In certainembodiments, the mature GAA polypeptide has a molecular weight ofapproximately 70 kilodaltons. In certain embodiments, the mature GAApolypeptide has a molecular weight of approximately 76 kilodaltons. Incertain embodiments, the mature GAA polypeptide consists of an aminoacid sequence selected from residues 122-782 of SEQ ID NO: 1 or residues204-782 of SEQ ID NO: 2. In certain embodiments, the chimericpolypeptide has acid alpha-glucosidase activity.

In certain embodiments, the disclosure provides for a method of treatingLafora Disease in a subject in need thereof, comprising administering achimeric polypeptide comprising: (i) a laforin polypeptide and (ii) aninternalizing moiety, such as an internalizing moiety that promotesdelivery into cells. In certain embodiments, the disclosure provides fora method of decreasing glycogen accumulation in cytoplasm of cells, suchas cells of a subject having Lafora Disease. In certain embodiments, themethod comprises contacting neuronal cells with a chimeric polypeptide,which chimeric polypeptide comprises (i) a laforin polypeptide and (ii)an internalizing moiety, such as an internalizing moiety that promotestransport into cytoplasm of cells. In certain embodiments, the subjector cells has a mutation in the EPM2A gene. In certain embodiments, thesubject or cells has a mutation in the EPM2B gene. In certainembodiments, the chimeric polypeptide for use in any of the methodsdisclosed herein comprises any of the laforin polypeptides describedherein. In certain embodiments, the laforin polypeptide comprises anamino acid sequence that is at least 80% identical to SEQ ID NO: 38 or39, or a biologically active fragment thereof. In certain embodiments,the laforin polypeptide comprises an amino acid sequence that is atleast 90% identical to SEQ ID NO: 38 or 39, or a biologically activefragment thereof. In certain embodiments, the laforin polypeptidecomprises an amino acid sequence that is at least 95% identical to SEQID NO: 38 or 39, or a biologically active fragment thereof.

In certain embodiments, the disclosure provides for a method of treatingAndersen Disease in a subject in need thereof, comprising administeringa chimeric polypeptide comprising: (i) an amyloglucosidase (AGL)polypeptide, and (ii) an internalizing moiety, such as an internalizingmoiety that promotes delivery into cells. In certain embodiments, thedisclosure provides for a method of decreasing glycogen accumulation incytoplasm of cells, such as cells of a subject having Andersen Disease.In certain embodiments, the method comprises administering a chimericpolypeptide, which chimeric polypeptide comprises (i) anamyloglucosidase (AGL) polypeptide, and (ii) an internalizing moiety,such as an internalizing moiety that promotes transport into cells. Incertain embodiments, the subject has a perinatal form of AndersenDisease. In certain embodiments, the subject has a congenital form ofAndersen Disease. In certain embodiments, the subject has a juvenileform of Andersen Disease. In certain embodiments, the subject has anadult form of Andersen Disease.

In certain embodiments, the disclosure provides for a method of treatingPompe Disease in a subject in need thereof, comprising administering achimeric polypeptide comprising: (i) an amyloglucosidase (AGL)polypeptide, and (ii) an internalizing moiety, such as an internalizingmoiety that promotes delivery into cells. In certain embodiments, thedisclosure provides for a method of decreasing glycogen accumulation incytoplasm of cells, such as cells of a subject having Pompe Disease. Incertain embodiments, the method comprises contacting muscle cells with achimeric polypeptide, which chimeric polypeptide comprises: (i) anamyloglucosidase (AGL) polypeptide, and (ii) an internalizing moiety,such as an internalizing moiety that promotes transport into cytoplasmof cells. In certain embodiments, the disclosure provides for a methodof treating von Gierke Disease in a subject in need thereof, comprisingadministering a chimeric polypeptide comprising: (i) an amyloglucosidase(AGL) polypeptide, and (ii) an internalizing moiety, such as aninternalizing moiety that promotes delivery into cells. In certainembodiments, the disclosure provides for a method of decreasing glycogenaccumulation in cytoplasm of cells, such as cells of a subject havingvon Gierke Disease. In certain embodiments, the method comprisescontacting liver cells with a chimeric polypeptide, which chimericpolypeptide comprises: (i) an amyloglucosidase (AGL) polypeptide, and(ii) an internalizing moiety, such as an internalizing moiety thatpromotes transport into cytoplasm of cells. In certain embodiments, thesubject or cells has a mutation in the gene encodingglucose-6-phosphatase. In certain embodiments, the subject or cells hasa mutation in the gene encoding SLC37A4.

In certain embodiments, the disclosure provides for a method of treatingLafora Disease in a subject in need thereof, comprising administering achimeric polypeptide comprising: (i) an amyloglucosidase (AGL)polypeptide, and (ii) an internalizing moiety, such as an internalizingmoiety that promotes delivery into cells. In certain embodiments, thedisclosure provides for a method of decreasing glycogen accumulation incytoplasm of cells, such as cells of a subject having Lafora Disease. Incertain embodiments, the method comprises contacting neuronal cells witha chimeric polypeptide, which chimeric polypeptide comprises: (i) anamyloglucosidase (AGL) polypeptide, and (ii) an internalizing moiety,such as an internalizing moiety that promotes transport into cytoplasmof cells. In certain embodiments, the subject or cells has a mutation inthe EPM2A gene. In certain embodiments,the subject has a mutation in theEPM2B gene.

In certain embodiments, the chimeric polypeptide for use in any of themethods disclosed herein comprises any of the AGL polypeptides describedherein. In certain embodiments, the AGL polypeptide for use in any ofthe methods disclosed herein comprises an amino acid sequence at least90%, 95%, 97% or 100% identical to any of SEQ ID NOs: 40-42. In certainembodiments, such AGL polypeptideshave amylo-1,6-glucosidase activityand 4-alpha-glucotransferase activity and the chimeric polypeptide hasamylo-1,6-glucosidase activity and 4-alpha-glucotransferase activity.

In certain embodiments, the disclosure provides for a method of treatingLafora Disease in a subject in need thereof, comprising administering achimeric polypeptide comprising: (i) a malin polypeptide, and (ii) aninternalizing moiety, such as an internalizing moiety that promotesdelivery into cells. In certain embodiments, the disclosure provides fora method of decreasing glycogen accumulation in cytoplasm of cells, suchas cells of a subject having Lafora Disease, comprising contactingneuronal cells with a chimeric polypeptide, which chimeric polypeptidecomprises (i) a malin polypeptide, and (ii) an internalizing moiety,such as an internalizing moiety that promotes transport into cytoplasmof cells. In certain embodiments, the subject or cells has a mutation inthe EPM2A gene. In certain embodiments, the subject or cells has amutation in the EPM2B gene.

In certain embodiments, the chimeric polypeptide for use in any of themethods disclosed herein comprises any of the malin polypeptidesdescribed herein. In certain embodiments, the malin polypeptide for usein any of the methods disclosed herein comprises an amino acid sequencethat is at least 80% identical to SEQ ID NO: 43, or a biologicallyactive fragment thereof. In certain embodiments, the malin polypeptidecomprises an amino acid sequence that is at least 90% identical to SEQID NO: 43, or a biologically active fragment thereof. In certainembodiments, the malin polypeptide comprises an amino acid sequence thatis at least 95% identical to SEQ ID NO: 43, or a biologically activefragment thereof.

In certain embodiments, the disclosure provides for a method of treatingLafora Disease in a subject in need thereof, comprising administering achimeric polypeptide comprising: (i) an alpha-amylase polypeptide, and(ii) an internalizing moiety, such as an internalizing moiety thatpromotes delivery into cells. In certain embodiments, the disclosureprovides for a method of decreasing glycogen accumulation in cytoplasmof cells, such as cells of a subject having Lafora Disease. In certainembodiments, the method comprises contacting neuronal cells with achimeric polypeptide, which chimeric polypeptide comprises (i) analpha-amylase polypeptide, and (ii) an internalizing moiety, such as aninternalizing moiety that promotes transport into cytoplasm of cells. Incertain embodiments, the subject or cells has a mutation in the EPM2Agene. In certain embodiments, the subject or cells has a mutation in theEPM2B gene.

In certain embodiments, the disclosure provides for a method of treatingForbes-Cori Disease in a subject in need thereof, comprisingadministering a chimeric polypeptide comprising: (i) an alpha-amylasepolypeptide, and (ii) an internalizing moiety. In certain embodiments,the internalizing moiety promotes delivery into cells, such as deliveryof the chimeric polypeptide. In certain embodiments, the disclosureprovides for a method of decreasing glycogen accumulation in cytoplasmof cells, such as cells of a subject having Forbes-Cori Disease. Incertain embodiments, the method comprises contacting neuronal cells witha chimeric polypeptide, which chimeric polypeptide comprises (i) analpha-amylase polypeptide, and (ii) an internalizing moiety. In certainembodiments, the internalizing moiety promotes transport into cytoplasmof cells. In certain embodiments, the subject or cell has a mutation inthe EPM2A gene. In certain embodiments, the subject or cell has amutation in the EPM2B gene.

In certain embodiments, the chimeric polypeptide for use in any of themethods disclosed herein comprises any of the alpha-amylase polypeptidesdescribed herein. In certain embodiments, the alpha-amylase polypeptidefor use in any of the methods disclosed herein comprises an amino acidsequence that is at least 80% identical to SEQ ID NO: 44 or 45, or abiologically active fragment thereof. In certain embodiments, thealpha-amylase polypeptide comprises an amino acid sequence that is atleast 90% identical to SEQ ID NO: 44 or 45, or a biologically activefragment thereof. In certain embodiments, the alpha-amylase polypeptidecomprises an amino acid sequence that is at least 95% identical to SEQID NO: 44 or 45, or a biologically active fragment thereof.

In certain embodiments, the two portions of the chimeric polypeptide maybe associated via any of a number of mechanisms (e.g., interconnectedvia one or more connections, such as one or more of chemicalconjugation, as a part of a fusion protein, disulfide bonds, etc).

In certain embodiments of any of the methods disclosed herein, theinternalizing moiety portion of the chimeric polypeptide promotesdelivery of the chimeric polypeptide into cells. In certain embodiments,the internalizing moiety promotes delivery of the chimeric polypeptideinto cytoplasm of cells. In certain embodiments, the chimericpolypeptide is capable of being taken up by an autophagic vacuole. Incertain embodiments, the internalizing moiety promotes delivery of saidchimeric polypeptide into muscle cells. In certain embodiments, theinternalizing moiety promotes delivery of said chimeric polypeptide intohepatocytes. In certain embodiments, the internalizing moiety promotestransport of said chimeric polypeptide into neurons. In certainembodiments, the chimeric polypeptide reduces cytoplasmic glycogenaccumulation. In certain embodiments, the internalizing moiety promotesdelivery of the chimeric polypeptide into cytoplasm of cells. In certainembodiments, the internalizing moiety comprises an antibody or antigenbinding fragment that can transit a cellular membrane via anequilibrative nucleoside transporter 2 (ENT2) transporter and/or bindsDNA with a K_(D) of less than 100 nM. In certain embodiments, theantibody is a monoclonal antibody or fragment thereof. In certainembodiments, the antibody or antigen binding fragment is a monoclonalantibody 3E10, or a variant thereof that retains cell penetratingactivity, or a variant thereof that binds the same epitope as 3E10, oran antibody that has substantially the same cell penetrating activity as3E10 and binds the same epitope as 3E10, or an antigen binding fragmentof any of the foregoing. In certain embodiments, the antibody or antigenbinding fragment is monoclonal antibody 3E10, or a variant thereof thatretains the cell penetrating activity of 3E10, or an antigen bindingfragment of 3E10 or said 3E10 variant. In certain embodiments, theinternalizing moiety comprises an antibody or antigen binding fragmentthat binds DNA (e.g., an anti-DNA antibody). In certain embodiments, theantibody or antigen binding fragment is a chimeric, humanized, or fullyhuman antibody or antigen binding fragment. In certain embodiments, theantibody or antigen binding fragment comprises a heavy chain variabledomain comprising an amino acid sequence at least 95% identical to SEQID NO: 9, or a humanized variant thereof. In certain embodiments, theantibody or antigen binding fragment comprises a light chain variabledomain comprising an amino acid sequence at least 95% identical to SEQID NO: 10, or a humanized variant thereof. In certain embodiments, theantibody or antigen binding fragment comprises a heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO: 9, or ahumanized variant thereof, and a light chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 10, or a humanized variantthereof. In certain embodiments, the antibody or antigen bindingfragment comprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO 13;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 14;

a VH CDR3 having the amino acid sequence of SEQ ID NO: 15;

a VL CDR1 having the amino acid sequence of SEQ ID NO: 16;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 17; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 18; which CDRsare according to Kabat.

In certain embodiments, the antibody or antigen binding fragmentcomprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO: 13;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 46; and

a VH CDR3 having the amino acid sequence of SEQ ID NO: 15,

a VL CDR1 having the amino acid sequence of SEQ ID NO: 16 or 47;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 48; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 18, which CDRsare according to Kabat.

In certain embodiments, the antibody or antigen binding fragmentcomprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO: 13;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 46; and

a VH CDR3 having the amino acid sequence of SEQ ID NO: 15,

a VL CDR1 having the amino acid sequence of SEQ ID NO: 16;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 48; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 18, which CDRsare according to Kabat.

In certain embodiments, the antibody or antigen binding fragmentcomprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO 24;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 25;

a VH CDR3 having the amino acid sequence of SEQ ID NO: 26;

a VL CDR1 having the amino acid sequence of SEQ ID NO: 27;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 28; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 29;

which CDRs are according to the IMGT system. In certain embodiments, theinternalizing moiety is an scFv. In certain embodiments, theinternalizing moiety is an Fab. In certain embodiments, theinternalizing moiety is an antibody. In some embodiments, theinternalizing moiety comprises a homing peptide. In some embodiments,the internalizing moiety is capable of binding DNA with a K_(D) of lessthan 100 nM. In some embodiments, the internalizing moiety binds DNAwith a K_(D) of less than 50 nM.

In certain embodiments, the chimeric polypeptide for use in any of themethods disclosed herein comprises N-linked oligosaccharide chainsmodified with M6P residues. In certain embodiments, the chimericpolypeptide further comprises one or more polypeptide portions thatenhance one or more of in vivo stability, in vivo half life,uptake/administration, production, or purification. In some embodiments,internalizing moiety transits cellular membranes via an equilibrativenucleoside transporter 1 (ENT1), ENT2, ENT3, or ENT4 transporter. Insome embodiments, the internalizing moiety can transit cellularmembranes via an equilibrative nucleoside transporter 2 (ENT2)transporter. In some embodiments, the chimeric polypeptide comprises afusion protein. In some embodiments, the chimeric polypeptide isproduced in a prokaryotic or eukaryotic cell. In some embodiments, theeukaryotic cell is selected from a yeast cell, an avian cell, an insectcell, or a mammalian cell.

In some embodiments, if the chimeric polypeptide comprises a GAApolypeptide, the C-terminus of the heavy chain of the Fab is fused tothe N-terminus of a GAA polypeptide. In some embodiments, the C-terminusof the heavy chain of the antibody is fused to the N-terminus of a GAApolypeptide. In some embodiments, the C-terminus of the heavy chain ofthe Fab is fused to the N-terminus of the GAA polypeptide by means of alinker. In some embodiments, the C-terminus of the heavy chain of theantibody is fused to the N-terminus of the GAA polypeptide by means of alinker. In some embodiments, the linker comprises the amino acidsequence of SEQ ID NO: 30. In some embodiments, the chimeric polypeptideis a chemical conjugate of GAA polypeptide to the internalizing moiety.In some embodiments, the chimeric polypeptide is a recombinant,co-translational fusion protein comprising the GAA polypeptide and theinternalizing moiety. In some embodiments, chimeric polypeptidecomprises a GAA polypeptide, and the GAA polypeptide is glycosylated. Incertain embodiments, the GAA polypeptide is not glycosylated. In certainembodiments, the GAA polypeptide has a glycosylation pattern thatdiffers from that of naturally occurring human GAA. In some embodiments,the chimeric polypeptide comprises a linker that conjugates or joins,directly or indirectly, the GAA polypeptide to the internalizing moiety.In some embodiments, the chimeric polypeptide does not include a linkerinterconnecting the GAA polypeptide to the internalizing moiety. In someembodiments, the linker is a cleavable linker. In some embodiments, theinternalizing moiety is N-terminal to the GAA polypeptide. In someembodiments, the internalizing moiety is conjugated or joined to aninternal amino acid of the GAA polypeptide. In some embodiments, thechimeric polypeptide has acid alpha-glucosidase activity, and whereinthe chimeric polypeptide does not comprise a GAA precursor polypeptideof approximately 110 kilodaltons.

In some embodiments, if the chimeric polypeptide comprises a laforinpolypeptide, the C-terminus of the heavy chain of the Fab is fused tothe N-terminus of a laforin polypeptide. In some embodiments, theC-terminus of the heavy chain of the antibody is fused to the N-terminusof a laforin polypeptide. In some embodiments, the C-terminus of theheavy chain of the Fab is fused to the N-terminus of the laforinpolypeptide by means of a linker. In some embodiments, the C-terminus ofthe heavy chain of the antibody is fused to the N-terminus of thelaforin polypeptide by means of a linker. In some embodiments, thelinker comprises the amino acid sequence of SEQ ID NO: 30. In someembodiments, wherein the chimeric polypeptide is a chemical conjugate oflaforin polypeptide and the internalizing moiety. In some embodiments,wherein the chimeric polypeptide is a recombinant, co-translationalfusion protein comprising the laforin polypeptide and the internalizingmoiety. In some embodiments, the chimeric polypeptide comprises a linkerthat conjugates or joins, directly or indirectly, the laforinpolypeptide to the internalizing moiety. In some embodiments,thechimeric polypeptide does not include a linker interconnecting thelaforin polypeptide to the internalizing moiety. In some embodiments,thelinker is a cleavable linker. In some embodiments,the internalizingmoiety is N-terminal to the laforin polypeptide. In some embodiments,the internalizing moiety is conjugated or joined to an internal aminoacid of the laforin polypeptide.

In some embodiments, if the chimeric polypeptide comprises an AGLpolypeptide, the C-terminus of the heavy chain of the Fab is fused tothe N-terminus of an AGL polypeptide. In some embodiments, theC-terminus of the heavy chain of the antibody is fused to the N-terminusof an AGL polypeptide. In some embodiments, the C-terminus of the heavychain of the Fab is fused to the N-terminus of the AGL polypeptide bymeans of a linker. In some embodiments, the C-terminus of the heavychain of the antibody is fused to the N-terminus of the AGL polypeptideby means of a linker. In some embodiments, the linker comprises theamino acid sequence of SEQ ID NO: 30. In some embodiments, the chimericpolypeptide is a chemical conjugate of AGL polypeptide and theinternalizing moiety. In some embodiments, the chimeric polypeptide is arecombinant, co-translational fusion protein comprising the AGLpolypeptide and the internalizing moiety. In some embodiments, thechimeric polypeptide comprises a linker that conjugates or joins,directly or indirectly, the AGL polypeptide to the internalizing moiety.In some embodiments, the chimeric polypeptide does not include a linkerinterconnecting the AGL polypeptide to the internalizing moiety. In someembodiments,the linker is a cleavable linker. In some embodiments, theinternalizing moiety is N-terminal to the AGL polypeptide. In someembodiments, the internalizing moiety is conjugated or joined to aninternal amino acid of the AGL polypeptide.

In some embodiments, if the chimeric polypeptide comprises a malinpolypeptide, the C-terminus of the heavy chain of the Fab is fused tothe N-terminus of a malin polypeptide. In some embodiments, theC-terminus of the heavy chain of the antibody is fused to the N-terminusof a malin polypeptide. In some embodiments, the C-terminus of the heavychain of the Fab is fused to the N-terminus of the malin polypeptide bymeans of a linker. In some embodiments, the C-terminus of the heavychain of the antibody is fused to the N-terminus of the malinpolypeptide by means of a linker. In some embodiments, the linkercomprises the amino acid sequence of SEQ ID NO: 30. In some embodiments,the chimeric polypeptide is a chemical conjugate of malin polypeptideand the internalizing moiety. In some embodiments, wherein the chimericpolypeptide is a recombinant, co-translational fusion protein comprisingthe malin polypeptide and the internalizing moiety. In some embodiments,the chimeric polypeptide comprises a linker that conjugates or joins,directly or indirectly, the malin polypeptide to the internalizingmoiety. In some embodiments, the chimeric polypeptide does not include alinker interconnecting the malin polypeptide to the internalizingmoiety. In some embodiments, the linker is a cleavable linker. In someembodiments, the internalizing moiety is N-terminal to the malinpolypeptide. In some embodiments, the internalizing moiety is conjugatedor joined to an internal amino acid of the malin polypeptide.

In some embodiments, if the chimeric polypeptide comprises analpha-amylase polypeptide, the C-terminus of the heavy chain of the Fabis fused to the N-terminus of an alpha-amylase polypeptide. In someembodiments, the C-terminus of the heavy chain of the antibody is fusedto the N-terminus of an alpha-amylase polypeptide. In some embodiments,the C-terminus of the heavy chain of the Fab is fused to the N-terminusof the alpha-amylase polypeptide by means of a linker. In someembodiments, the C-terminus of the heavy chain of the antibody is fusedto the N-terminus of the alpha-amylase polypeptide by means of a linker.In some embodiments, the linker comprises the amino acid sequence of SEQID NO: 30. In some embodiments, the chimeric polypeptide is a chemicalconjugate of alpha-amylase polypeptide and the internalizing moiety. Insome embodiments, the chimeric polypeptide is a recombinant,co-translational fusion protein comprising the alpha-amylase polypeptideand the internalizing moiety. In some embodiments, the chimericpolypeptide comprises a linker that conjugates or joins, directly orindirectly, the alpha-amylase polypeptide to the internalizing moiety.In some embodiments, the chimeric polypeptide does not include a linkerinterconnecting the alpha-amylase polypeptide to the internalizingmoiety. In some embodiments, the linker is a cleavable linker. In someembodiments, the internalizing moiety is N-terminal to the alpha-amylasepolypeptide. In some embodiments, the internalizing moiety is conjugatedor joined to an internal amino acid of the alpha-amylase polypeptide.

In some embodiments, any of the chimeric polypeptides disclosed hereinhas acid glucosidase activity.

In some embodiments of any of the methods described herein, the chimericpolypeptide is administered parenterally. In some embodiments, thechimeric polypeptide is administered intravenously. In some embodiments,the chimeric polypeptide is administered intramuscularly. In someembodiments, the chimeric polypeptide is administered subcutaneously. Insome embodiments, the chimeric polypeptide is administered intravenouslyvia bolus injection or infusion. In some embodiments, the chimericpolypeptide is administered via the hepatic portal vein. In someembodiments, wherein the chimeric polypeptide is administeredintracranially or intrathecally.

In some embodiments of any of the methods disclosed herein compriseadministering an effective amount of the chimeric polypeptide. In someembodiments, the method decreases or clears glycogen accumulation, andthe glycogen comprises polyglucosan.

In some embodiments, the disclosure provides for a compositioncomprising a chimeric polypeptide formulated with one or morepharmaceutically acceptable carriers and/or excipients, which chimericpolypeptide comprises (i) an acid alpha-glucosidase (GAA) polypeptide(e.g., a GAA polypeptide comprising mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cells, wherein at least 90% of the GAA polypeptidepresent in the composition is interconnected to an internalizing moiety.In some embodiments, at least 95% of the GAA polypeptide present in thecomposition is interconnected to an internalizing moiety. In someembodiments, at least 96% or at least 97% of the GAA polypeptide presentin the composition is interconnected to an internalizing moiety.

In some embodiments, the disclosure provides for a compositioncomprising a chimeric polypeptide formulated with one or morepharmaceutically acceptable carriers and/or excipients, which chimericpolypeptide comprises (i) an acid alpha-glucosidase (GAA) polypeptide(e.g., a GAA polypeptide comprising mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cells, wherein greater than 85% of the GAA polypeptidepresent in the composition has substantially the same amino acidsequence. In some embodiments, at least 90% of the GAA polypeptidepresent in the composition has substantially the same amino acidsequence. In some embodiments, greater than 90% of the GAA polypeptidepresent in the composition has the same interconnection to aninternalizing moiety. In some embodiments, at least 95% of the GAApolypeptide present in the composition is interconnected to aninternalizing moiety. In some embodiments, greater than 85% of the GAApolypeptide present in the composition is approximately the samemolecular weight. In some embodiments, greater than 90% of the GAApolypeptide present in the composition differs at the N-terminus of aGAA polypeptide portion by less than 5, 4, 3, 2, or 1 residues.

In some embodiments, any of the compositions disclosed herein comprisinga chimeric polypeptide that comprises a GAA polypeptide aresubstantially free of mature GAA that does not include additionalcontiguous GAA sequence and/or that is not interconnected to aninternalizing moiety. In some embodiments, the composition comprisesless than 5% by weight of mature GAA that does not include additionalcontiguous GAA sequence and/or that is not interconnected to aninternalizing moiety.

In some embodiments, the disclosure provides for a compositioncomprising chimeric polypeptides formulated with one or morepharmaceutically acceptable carriers and/or excipients, which chimericpolypeptides comprise (i) an acid alpha-glucosidase (GAA) polypeptide(e.g., a GAA polypeptide comprising mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cells, wherein at least 85% of the chimeric polypeptidesin the composition comprise an amino acid sequence that differs by lessthan 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues. In someembodiments, at least 90% of the chimeric polypeptides in thecomposition comprise an amino acid sequence that differs by less than10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues. In someembodiments, at least 95% of the chimeric polypeptides in thecomposition comprise an amino acid sequence that differs by less than 5,4, 3, 2, or 1 amino acid residues.

In some embodiments, the disclosure provides for a compositioncomprising chimeric polypeptides formulated with one or morepharmaceutically acceptable carriers and/or excipients, which chimericpolypeptides comprise (i) an acid alpha-glucosidase (GAA) polypeptide(e.g., a GAA polypeptide comprising mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cells, wherein at least 85% of the GAA present in thecomposition comprises an amino acid sequence that differs by less than10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues. In someembodiments, at least 90% of the GAA in the composition comprise anamino acid sequence that differs by less than 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 amino acid residues. In some embodiments, at least 95% of theGAA in the composition comprise an amino acid sequence that differs byless than 5, 4, 3, 2, or 1 amino acid residues.

In some embodiments, the disclosure provides for a compositioncomprising a chimeric polypeptide formulated with one or morepharmaceutically acceptable carriers and/or excipients, which chimericpolypeptide comprises (i) an acid alpha-glucosidase (GAA) polypeptide(e.g., a GAA polypeptide comprising mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cells, wherein the composition is substantially free ofmature GAA that does not include additional contiguous GAA sequenceand/or that is not interconnected to an internalizing moiety.

In some embodiments, the disclosure provides for a compositioncomprising chimeric polypeptides formulated with one or morepharmaceutically acceptable carriers and/or excipients, which chimericpolypeptides comprise (i) an acid alpha-glucosidase (GAA) polypeptide(e.g., a GAA polypeptide comprising mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cytoplasm of cells, wherein at least 91% of the GAApolypeptide present in the composition is interconnected to aninternalizing moiety. In certain embodiments, at least 95% of the GAApolypeptide in the composition is interconnected to an internalizingmoiety. In certain embodiments, at least 98% of the GAA polypeptide inthe composition is interconnected to the internalizing moiety.

In some embodiments, the disclosure provides for a compositioncomprising a chimeric polypeptide formulated with one or morepharmaceutically acceptable carriers and/or excipients, which chimericpolypeptide comprises (i) an acid alpha-glucosidase (GAA) polypeptide(e.g., a GAA polypeptide comprising mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cells, wherein at least 85% of the chimeric polypeptidespresent in the composition have the same amino acid sequence. In someembodiments, at least 90% of the chimeric polypeptides in thecomposition have the same amino acid sequence. In some embodiments, atleast 95% of the chimeric polypeptides in the composition have the sameamino acid sequence.

In some embodiments, the disclosure provides for a compositioncomprising a chimeric polypeptide formulated with one or morepharmaceutically acceptable carriers and/or excipients, which chimericpolypeptide comprises (i) an acid alpha-glucosidase (GAA) polypeptide(e.g., a GAA polypeptide comprising mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cells, wherein at least 85% of the GAA present in thecomposition has the same amino acid sequence. In certain embodiments, atleast 90% of the GAA present in the composition has the same amino acidsequence. In certain embodiments, at least 95% of the GAA present in thecomposition has the same amino acid sequence.

In some embodiments, the disclosure provides for a compositioncomprising a chimeric polypeptide formulated with one or morepharmaceutically acceptable carriers and/or excipients, which chimericpolypeptide comprises (i) an acid alpha-glucosidase (GAA) polypeptide(e.g., a GAA polypeptide comprising mature GAA) and (ii) aninternalizing moiety, such as an internalizing moiety that promotestransport into cells, wherein less than 10% of the GAA present in thecomposition is a mature GAA polypeptide. In some embodiments, less than5% of the GAA present in the composition is a mature GAA polypeptide. Insome embodiments, less than 2% of the GAA present in the composition isa mature GAA polypeptide. In some embodiments, the GAA polypeptidecomprises the amino acid sequence of SEQ ID NO: 22. In some embodiments,the chimeric polypeptide comprises an immunoglobulin or epitope tag. Insome embodiments, the immunoglobulin or epitope tag is used forpurification of the chimeric polypeptide.

In certain embodiments, any of the compositions described hereincomprises any of the chimeric polypeptides disclosed herein. In certainembodiments, the chimeric polypeptides comprise any of the GAApolypeptides disclosed herein. In certain embodiments, the chimericpolypeptides comprise any of the internalizing moieties disclosedherein.

In certain embodiments, any of the compositions disclosed herein issubstantially pyrogen free. In certain embodiments, the composition isin a bottle. In certain embodiments, the composition is in a syringe. Incertain embodiments, the composition is stored prior to administration.In certain embodiments, any of the compositions dislosed herein may beused for treating one or more of Pompe Disease, Forbes Cori Disease,Andersen Disease, von Gierke Disease or Lafora Disease. In certainembodiments, any of the compositions disclosed herein may be used in amethod for delivering GAA activity into cells. In some embodiments, theGAA activity is delivered to cytoplasm of the cells. In someembodiments, the cell is in vitro, and wherein the cell in vitro is froma subject having Forbes Cori Disease, Andersen Disease, Pompe Disease,Lafora Disease or von Gierke Disease. In some embodiments, the cell isin a subject, and wherein the subject has Forbes Cori Disease, AndersenDisease, Pompe Disease, Lafora Disease or von Gierke Disease.

In some embodiments, the disclosure provides for a chimeric polypeptidecomprising: (i) a laforin polypeptide, and (ii) an internalizing moiety(e.g., any of the internalizing moieties described herein). In someembodiments, the laforin polypeptide comprises an amino acid sequence atleast 90% identical to SEQ ID NO: 38 or 39, or a biologically activefragment thereof. In some embodiments, the laforin polypeptide comprisesan amino acid sequence at least 95% identical to SEQ ID NO: 38 or 39, ora biologically active fragment thereof. In some embodiments, thechimeric polypeptide has glucan phosphatase activity. In someembodiments, the chimeric polypeptide has protein phosphatase activity.In some embodiments, the chimeric polypeptide is capable of bindingcarbohydrates. In some embodiments, the chimeric polypeptide is capableof forming a complex with malin. In some embodiments, the laforinpolypeptide is chemically conjugated to the internalizing moiety. Insome embodiments, the chimeric polypeptide comprises a fusion proteincomprising the laforin polypeptide and the internalizing moiety. In someembodiments, the chimeric polypeptide comprises a fusion protein. Insome embodiments, the fusion protein comprises a linker. In someembodiments, the chimeric polypeptide comprises a linker. In someembodiments, the linker conjugates or joins the malin polypeptide to theinternalizing moiety. In some embodiments, the chimeric polypeptide doesnot include a linker interconnecting the laforin polypeptide to theinternalizing moiety. In some embodiments, the linker is a cleavablelinker. In some embodiments, the internalizing moiety is conjugated orjoined, directly or indirectly, to the N-terminal or C-terminal aminoacid of the laforin polypeptide. In some embodiments, the internalizingmoiety is conjugated or joined, directly or indirectly to an internalamino acid of the laforin polypeptide.

In some embodiments, the disclosure provides for a chimeric polypeptidecomprising: (i) a malin polypeptide, and (ii) an internalizing moiety(e.g., any of the internalizing moieties described herein). In someembodiments, the malin polypeptide comprises an amino acid sequence atleast 90% identical to SEQ ID NO: 43, or a biologically active fragmentthereof. In some embodiments, the malin polypeptide comprises an aminoacid sequence at least 95% identical to SEQ ID NO: 43, or a biologicallyactive fragment thereof. In some embodiments, the chimeric polypeptidehas E3 ubiquitin ligase activity. In some embodiments, the chimericpolypeptide is capable of forming a complex with laforin. In someembodiments, the malin polypeptide is chemically conjugated to theinternalizing moiety. In some embodiments, the chimeric polypeptidecomprises a fusion protein comprising the malin polypeptide and theinternalizing moiety. In some embodiments, the chimeric polypeptidecomprises a fusion protein. In some embodiments, the fusion proteincomprises a linker. In some embodiments, the chimeric polypeptidecomprises a linker. In some embodiments, the linker conjugates or joinsthe malin polypeptide to the internalizing moiety. In some embodiments,the chimeric polypeptide does not include a linker interconnecting themalin polypeptide to the internalizing moiety. In some embodiments, thelinker is a cleavable linker. In some embodiments, the internalizingmoiety is conjugated or joined, directly or indirectly, to theN-terminal or C-terminal amino acid of the malin polypeptide. In someembodiments, the internalizing moiety is conjugated or joined, directlyor indirectly to an internal amino acid of the malin polypeptide.

In some embodiments, the disclosure provides for a chimeric polypeptidecomprising: (i) an alpha-amylase polypeptide, and (ii) an internalizingmoiety (e.g., any of the internalizing moieties described herein). Insome embodiments, the alpha-amylase polypeptide is a pancreaticalpha-amylase. In some embodiments, the alpha-amylase polypeptide is asalivary alpha-amylase. In some embodiments, the alpha-amylasepolypeptide comprises an amino acid sequence at least 90% identical toSEQ ID NO: 44 or 45, or a biologically active fragment thereof. In someembodiments, the alpha-amylase polypeptide comprises an amino acidsequence at least 95% identical to SEQ ID NO: 44 or 45, or abiologically active fragment thereof. In some embodiments, the chimericpolypeptide has alpha-1,4-glucosidic bonds hydrolytic activity. In someembodiments, the alpha-amylase polypeptide is chemically conjugated tothe internalizing moiety. In some embodiments, the chimeric polypeptidecomprises a fusion protein comprising the alpha-amylase polypeptide andthe internalizing moiety. In some embodiments, the chimeric polypeptidecomprises a fusion protein. In some embodiments, the fusion proteincomprises a linker. In some embodiments, the chimeric polypeptidecomprises a linker. In some embodiments, the linker conjugates or joinsthe alpha-amylase polypeptide to the internalizing moiety. In someembodiments, the chimeric polypeptide does not include a linkerinterconnecting the alpha-amylase polypeptide to the internalizingmoiety. In some embodiments, the linker is a cleavable linker. In someembodiments, the internalizing moiety is conjugated or joined, directlyor indirectly, to the N-terminal or C-terminal amino acid of thealpha-amylase polypeptide. In some embodiments,the internalizing moietyis conjugated or joined, directly or indirectly to an internal aminoacid of the alpha-amylase polypeptide.

In certain embodiments of any of the methods or compositions disclosedherein, the internalizing moiety portion of the chimeric polypeptidepromotes delivery of the chimeric polypeptide into cells. In certainembodiments, the internalizing moiety promotes delivery of the chimericpolypeptide into cytoplasm of cells. In certain embodiments, thechimeric polypeptide is capable of being taken up by an autophagicvacuole. In certain embodiments, the internalizing moiety promotesdelivery of said chimeric polypeptide into muscle cells. In certainembodiments, the internalizing moiety promotes delivery of said chimericpolypeptide into hepatocytes. In certain embodiments, the internalizingmoiety promotes transport of said chimeric polypeptide into neurons. Incertain embodiments, the chimeric polypeptide reduces cytoplasmicglycogen accumulation. In certain embodiments, the internalizing moietypromotes delivery of the chimeric polypeptide into cytoplasm of cells.In certain embodiments, the internalizing moiety comprises an antibodyor antigen binding fragment that can transit a cellular membrane via anequilibrative nucleoside transporter 2 (ENT2) transporter and/or bindsDNA with a K_(D) of less than 100 nM. In certain embodiments, theantibody is a monoclonal antibody or fragment thereof. In certainembodiments, the antibody or antigen binding fragment is a monoclonalantibody 3E10, or a variant thereof that retains cell penetratingactivity, or a variant thereof that binds the same epitope as 3E10, oran antibody that has substantially the same cell penetrating activity as3E10 and binds the same epitope as 3E10, or an antigen binding fragmentof any of the foregoing. In certain embodiments, the antibody or antigenbinding fragment is monoclonal antibody 3E10, or a variant thereof thatretains the cell penetrating activity of 3E10, or an antigen bindingfragment of 3E10 or said 3E10 variant. In certain embodiments, theinternalizing moiety comprises an antibody or antigen binding fragmentthat binds DNA (e.g., an anti-DNA antibody). In certain embodiments, theantibody or antigen binding fragment is a chimeric, humanized, or fullyhuman antibody or antigen binding fragment. In certain embodiments, theantibody or antigen binding fragment comprises a heavy chain variabledomain comprising an amino acid sequence at least 95% identical to SEQID NO: 9, or a humanized variant thereof.

In certain embodiments, the antibody or antigen binding fragmentcomprises a light chain variable domain comprising an amino acidsequence at least 95% identical to SEQ ID NO: 10, or a humanized variantthereof. In certain embodiments, the antibody or antigen bindingfragment comprises a heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 9, or a humanized variant thereof, and alight chain variable domain comprising the amino acid sequence of SEQ IDNO: 10, or a humanized variant thereof. In certain embodiments, theantibody or antigen binding fragment comprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO 13;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 14;

a VH CDR3 having the amino acid sequence of SEQ ID NO: 15;

a VL CDR1 having the amino acid sequence of SEQ ID NO: 16;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 17; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 18; which CDRsare according to Kabat.

In certain embodiments, the antibody or antigen binding fragmentcomprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO: 13;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 46; and

a VH CDR3 having the amino acid sequence of SEQ ID NO: 15,

a VL CDR1 having the amino acid sequence of SEQ ID NO: 16 or 47;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 48; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 18, which CDRsare according to Kabat.

In certain embodiments, the antibody or antigen binding fragmentcomprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO: 13;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 46; and

a VH CDR3 having the amino acid sequence of SEQ ID NO: 15,

a VL CDR1 having the amino acid sequence of SEQ ID NO: 16;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 48; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 18, which CDRsare according to Kabat.

In certain embodiments, the antibody or antigen binding fragmentcomprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO 24;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 25;

a VH CDR3 having the amino acid sequence of SEQ ID NO: 26;

a VL CDR1 having the amino acid sequence of SEQ ID NO: 27;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 28; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 29;

which CDRs are according to the IMGT system. In certain embodiments, theinternalizing moiety is an scFv. In certain embodiments, theinternalizing moiety is an Fab.

In certain embodiments, the internalizing moiety is an antibody. In someembodiments, the internalizing moiety comprises a homing peptide. Insome embodiments, the internalizing moiety is capable of binding DNAwith a K_(D) of less than 100 nM. In some embodiments, the internalizingmoiety binds DNA with a K_(D) of less than 50 nM. In certainembodiments, the chimeric polypeptide for use in any of the methodsdisclosed herein comprises N-linked oligosaccharide chains modified withM6P residues. In certain embodiments, the chimeric polypeptide furthercomprises one or more polypeptide portions that enhance one or more ofin vivo stability, in vivo half life, uptake/administration, production,or purification. In some embodiments, internalizing moiety transitscellular membranes via an equilibrative nucleoside transporter 1 (ENT1),ENT2, ENT3, or ENT4 transporter. In some embodiments, the internalizingmoiety can transit cellular membranes via an equilibrative nucleosidetransporter 2 (ENT2) transporter. In some embodiments, the chimericpolypeptide comprises a fusion protein. In some embodiments, thechimeric polypeptide is produced in a prokaryotic or eukaryotic cell. Insome embodiments, the eukaryotic cell is selected from a yeast cell, anavian cell, an insect cell, or a mammalian cell.

In certain embodiments, the disclosure provides for a nucleic acidconstruct, comprising a nucleotide sequence that encodes any of thechimeric polypeptides disclosed herein as a fusion protein. In someembodiments, the disclosure provides for a vector comprising any of thenucleic acids disclosed herein. In some embodiments, the disclosureprovides for a host cell comprising any of the vectors disclosed herein.

In some embodiments, the disclosure provides for a method of deliveringactivity into cells, comprising contacting cells with any of thechimeric polypeptides disclosed herein. In some embodiments, the cell isin vitro from a subject having Forbes Cori Disease, Andersen Disease,Pompe Disease, Lafora Disease or von Gierke Disease. In someembodiments, the cell is in a subject, and wherein the subject hasForbes Cori Disease, Andersen Disease, Pompe Disease, Lafora Disease orvon Gierke Disease. In some embodiments, the subject has Lafora Disease.In some embodiments, the disclosure provides for a method for decreasingglycogen accumulation, such as in a cell or in a subject in needthereof, comprising administering any the chimeric polypeptidesdisclosed herein. In some embodiments, the subject in need thereof hasForbes Cori Disease, Andersen Disease, Pompe Disease, Lafora Disease orvon Gierke Disease. In some embodiments, the subject in need thereof hasLafora Disease.

The disclosure contemplates that any one or more of the aspects andembodiments of the disclosure detailed above can be combined with eachother and/or with any of the features disclosed below. Moreover, any oneor more of the features of the disclosure described below may becombined.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1C provide a diagram schematically depicting two differentfusion constructs generated. FIG. lA is a diagram schematicallydepicting the full-length GAA protein and its different regions, as wellas the murine heavy and light chains of an internalizing moiety, in thiscase, a DNA binding antibody. Amino acid residues 1-28 correspond to thesignal sequence (“SigSeq”) region, amino acids 29-56 correspond to theprepro region, and amino acids 57-78 corresponds to the full linkerregion. Residues 1-56 are highlighted in SEQ ID NO: 1 because, inaccordance with Moreland et al., this is the portion of the GAAtranslation product that is cleaved by a signal peptidase and proteaseto produce the precursor GAA polypeptide of approximately 110kilodaltons. FIG. 1B is a diagram schematically depicting the murine3E10 Fab-human GAA fusion construct, while FIG. 1C is a diagramschematically depicting the murine 3E10 mAb-human GAA fusion construct.These are exemplary of chimeric polypeptides comprising a GAApolypeptide and internalizing moiety, in accordance with the disclosure.These include examples in which the internalizing moiety is a fulllength antibody and examples in which the internalizing moiety is a Fab.

FIG. 2 depicts representative SEC-HPLC charts for purified humanizedFab-GAA. FIGS. 3A-3B show representative gels resulting from SDS-PAGEanalysis of Fab-GAA protein, as detected using an anti-GAA monoclonalantibody (Sigma, SAB2100872). Fab-GAA was detected in either medium(FIG. 3A) or cell lysates (FIG. 3B) from Pompe fibroblast culturestreated either with (T1 and T2) or without (C1 and C2) humanized 3E10Fab-GAA. Beta-actin levels were assessed as a loading control for thecell lysate samples.

FIG. 4 shows a representative gel resulting from SDS-PAGE analysis ofGAA protein as detected using an anti-GAA monoclonal antibody (Sigma,SAB2100872). GAA was detected in L6 rat skeletal muscle cells treatedwith or without (control) humanized or murine 3E10 Fab-GAA (hFab-GAA andmFab-GAA, respectively) in the presence or absence ofmannose-6-phosphate (M6P). ENT2 and M6P-Receptor (M6PR) levels were alsomeasured using an anti-ENT2 antibody (Santa Cruz, sc-134569) and ananti-M6PR antibody (Abcam, ab124767), respectively. Beta-actin levelswere assessed as a loading control.

DETAILED DESCRIPTION OF THE DISCLOSURE

Glycogen is a complex polysaccharide that provides a ready store ofglucose to cells in the human body. Glycogen is found principally in theliver, where it is hydrolyzed and released into the bloodstream toprovide glucose to other cells, and in muscle, where the glucoseresulting from glycogen hydrolysis provides energy for muscle cells. Theenzymes acid a-glucosidase (GAA), alpha-amylase and amyloglucosidase(AGL) are some of the enzymes that mediate glycogen hydrolysis. Laforinand malin are also believed to play a role in glycogen clearance.

Disruption of glycogen hydrolysis, typically resulting from geneticmutations in genes associated with the process, can lead to glycogenstorage disorders. In many cases, the severity of the disease symptomscorrelates directly with the extent of the mutation.

The art does not describe any reliable means of administering GAA,laforin, alpha-amylase, malin and/or AGL to Forbes-Cori, Andersen, vonGierke, and/or Lafora Disease cells such that the GAA, laforin,alpha-amylase, malin and/or AGL would be internalized in such a way asto clear, for example, cytoplasmic accumulation of glycogen. As such, inone embodiment, the disclosure provides for chimeric polypeptidescomprising: a) a GAA polypeptide comprising the amino acid sequence ofSEQ ID NOs: 1 or 2, or a fragment thereof comprising mature GAA, and b)an internalizing moiety that delivers the GAA polypeptide to thecytoplasm of a cell. The disclosure also provides for chimericpolypeptides comprising: a) a laforin polypeptide comprising the aminoacid sequence of SEQ ID NOs: 38 or 39, or biologically active fragmentthereof, and b) an internalizing moiety that delivers the laforinpolypeptide into cells, such as into the cytoplasm of a cell. Thedisclosure also provides for chimeric polypeptides comprising: a) an AGLpolypeptide comprising the amino acid sequence of any of SEQ ID NOs:40-42, or biologically active fragment thereof, and b) an internalizingmoiety that delivers the AGL polypeptide to cells, such as into thecytoplasm of a cell. The disclosure also provides for chimericpolypeptides comprising: a) a malin polypeptide comprising the aminoacid sequence of SEQ ID NO: 43, or biologically active fragment thereof,and b) an internalizing moiety that delivers the malin polypeptide intocells, such as into the cytoplasm of a cell. The disclosure alsoprovides for chimeric polypeptides comprising: a) an alpha-amylasepolypeptide comprising the amino acid sequence of SEQ ID NO: 44 or 45,or biologically active fragment thereof, and b) an internalizing moietythat delivers the alpha-amylase polypeptide into cells, such as into thecytoplasm of a cell.

Similar to Forbes-Cori, Andersen Disease and Lafora Disease are alsoassociated with cytoplasmic accumulation of glycogen (Magoulas P L andEl-Hattab A W, 2013, Gene Reviews; Gentry et al., 2013, FEBS J.,280(2):525-37). As such, the chimeric polypeptides disclosed hereinwould have similar utility in clearing cytoplasmic glycogen accumulationin Andersen Disease or Lafora Disease cells. Moreover, a chimericpolypeptide, as provided herein, is useful for clearing cytoplasmicglycogen accumulation in von Gierke Disease cells and/or Pompe Diseasecells.

Such cells include cells in culture, such as cells from a patient havingone of these diseases or from an animal model of one of these diseases,as well as cells in a patient having one of these glycogen storage ormetabolism disorders.

The present disclosure provides chimeric polypeptides and compositionsand various methods of using such chimeric polypeptides. The chimericpolypeptides of the disclosure include an internalizing moiety portionthat promote delivery into cells and a non-internalizing moietypolypeptide portion. The internalizing moiety portion and thenon-internalizing moiety polypeptide portion are associated, such asconjugated or otherwise joined. In certain embodiments, thenon-internalizing moiety polypeptide portion of the chimeric polypeptideis a GAA polypeptide, and numerous examples of GAA polypeptides for usein the methods and compositions of the disclosure are provided anddescribed in detail herein. In certain embodiments, thenon-internalizing moiety polypeptide portion of the chimeric polypeptideis an AGL polypeptide, and numerous examples of AGL polypeptides for usein the methods and compositions of the disclosure are provided anddescribed in detail herein. In certain embodiments, thenon-internalizing moiety polypeptide portion of the chimeric polypeptideis a laforin polypeptide, and numerous examples of laforin polypeptidesfor use in the methods and compositions of the disclosure are providedand described in detail herein. In certain embodiments, thenon-internalizing moiety polypeptide portion of the chimeric polypeptideis a malin polypeptide, and numerous examples of malin polypeptides foruse in the methods and compositions of the disclosure are provided anddescribed in detail herein. In certain embodiments, thenon-internalizing moiety polypeptide portion of the chimeric polypeptideis an alpha-amylase polypeptide, and numerous examples of alpha-amylasepolypeptides for use in the methods and compositions of the disclosureare provided and described in detail herein.

I. GAA Polypeptides

In certain embodiments, the chimeric polypeptides for use in the methodsdisclosed herein comprise a GAA polypeptide, e.g., a GAA polypeptidecomprising or consisting of mature GAA. It has been demonstrated thatmature GAA polypeptides have enhanced glycogen clearance as compared tothe full length, precursor GAA (Bijvoet, et al., 1998, Hum Mol Genet,7(11): 1815-24), whether at low pH (i.e., the pH of the lysosome orautophagic vacuole) or neutral pH (i.e., the pH of the cytoplasm)conditions. In addition, while mature GAA is a lysosomal protein thathas optimal activity at lower pHs, mature GAA retains approximately 40%activity at neutral pH (i.e., the pH of the cytoplasm) (Martin-Touaux etal., 2002, Hum Mol Genet, 11(14): 1637-45). Accordingly, a GAApolypeptide comprising mature GAA is suitable for cytoplasmic delivery,and thus, suitable to address an unaddressed issue of Forbes-Cori, vonGierke, Lafora and/or Andersen Disease: cytoplasmic glycogenaccumulation. However, regardless of whether the GAA portion of achimeric polypeptide comprises or consists of mature GAA, providing theGAA polypeptide in association with an internalizing moiety of thedisclosure facilitates delivery into cells and, in certain embodiments,delivery to cytoplasm. In certain embodiments, the chimeric polypeptideis capable of entering the cytoplasm of cells in the presence ofinhibitors of mannose-6-phophate receptors (MPRs). Without being boundby theory, administration of any of the chimeric polypeptides disclosedherein, such as a GAA polypeptide comprising mature GAA and aninternalizing moiety to a patient would ensure that mature GAA reachedtissues such as muscle and liver and that GAA activity was not limitedto the lysosome.

Without being bound by theory, the administered GAA polypeptide (e.g.,chimeric polypeptides of the disclosure comprising GAA) will act in, atleast, the cytoplasm to reduce the deleterious glycogen accumulationthat results from AGL mutations in Forbes-Cori patients; GAA mutationsin Pompe Disease; G6PC or SLC37A4 mutations in von Gierke Diseasepatients; EPM2A and/or EPM2B mutations in Lafora Disease; and/or GBEmutations in Andersen Disease patients. In some embodiments, theadministered GAA polypeptide will act in, at least, the lysosomes orvacuoles (e.g., autophagic vacuoles) to reduce the deleterious glycogenaccumulation that results from AGL mutations in Forbes-Cori patients;G6PC or SLC37A4 mutations in von Gierke Disease patients; EPM2A and/orEPM2B mutations in Lafora Disease; and/or GBE mutations in AndersenDisease patients. By reducing deleterious glycogen accumulation incells, particularly in muscle cells (e.g., skeletal and/or cardiacmuscle), neurons or glia (in some indications) and/or liver cells,delivery of GAA, laforin, alpha-amylase, malin and/or AGL activity tocytoplasm of cells in patients in need thereof is useful for alleviating some or all of the symptoms associated with glycogen accumulationin the patient's cells, including accumulation of abnormal glycogenaccumulation (e.g., polyglucosan). In some embodiments, the delivery ofGAA, laforin, alpha-amylase, malin and/or AGL activity to lysosomes orvacuoles (e.g., autophagic vacuoles) in patients in need thereof isuseful for alleviating some or all of the symptoms associated withglycogen accumulation in the patient's cells, including accumulation ofabnormal glycogen accumulation (e.g., polyglucosan). In certainembodiments, chimeric polypeptides of the disclosure deliver GAAactivity to cytoplasm and to one or both of lysosome and/or vacuoles(e.g., autophagic vacuoles). Accordingly, delivery of GAA, laforin,alpha-amylase, malin and/or AGL activity using chimeric polypeptides ofthe disclosure is suitable for treating Forbes-Cori Disease, PompeDisease, Andersen Disease, von Gierke Disease and/or Lafora Disease. Incertain embodiments, a chimeric polypeptide of the disclosure issuitable for treating Pompe Disease, Forbes-Cori Disease, AndersenDisease, Lafora Disease and/or von Gierke Disease, in a patient in needthereof.

In certain embodiments, a chimeric polypeptide comprising a GAApolypeptide and an internalizing moiety can enter into a cell, such asinto the cytoplasm, in the presence of an agent that blocksmannose-6-phophate receptors (MPRs).

In certain embodiments, the disclosure provides a chimeric polypeptidecomprising a GAA polypeptide and an internalizing moiety, as describedherein. Any such chimeric polypeptide of the disclosure can comprise anyof the GAA polypeptides described herein associated with any of theinternalizing moiety portions described herein, and these chimericpolypeptides can be used in any of the methods of the disclosure.

In certain embodiments, the disclosure provides a chimeric polypeptidecomprising a GAA polypeptide and an internalizing moiety, as describedherein. Any such chimeric polypeptide of the disclosure can comprise anyof the GAA polypeptides described herein associated with any of theinternalizing moiety portions described herein, and these chimericpolypeptides can be used in any of the methods of the disclosure.

In certain aspects, the disclosure provides using a GAA protein (e.g., aGAA polypeptide comprising a mature GAA protein) , laforin polypeptide,alpha-amylase polypeptide, malin polypeptide and/or an AGL polypeptideto treat conditions associated with aberrant accumulation of abnormalamounts and/or types of glycogen such as occurs in Forbes-Cori Disease,Pompe Disease, von Gierke Disease, Lafora Disease and/or AndersenDisease. The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

Thus, in certain aspects, the disclosure provides chimeric polypeptidescomprising an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising or consisting of mature GAA polypeptide), laforinpolypeptide, alpha-amylase polypeptide, malin polypeptide and/or an AGLpolypeptide that may be used to treat symptoms associated withForbes-Cori and/or Andersen Disease and/or Pompe Disease and/or vonGierke Disease and/or Lafora Disease. In certain embodiments, chimericpolypeptides of the disclosure have GAA, laforin, alpha-amylase, malinand/or AGL biological activity. For example, chimeric polypeptides ofthe disclosure comprise a GAA, laforin, alpha-amylase and/or AGLpolypeptide having enzymatic activity.

In certain embodiments, the disclosure provides a chimeric polypeptidecomprising (i) a GAA polypeptide (e.g., a GAA polypeptide comprising orconsisting of mature GAA polypeptide), laforin polypeptide,alpha-amylase polypeptide, malin polypeptide and/or an AGL polypeptide;and (ii) an internalizing moiety that promotes delivery into cells, suchas into cytoplasm of cells (e.g., into cytoplasm of muscle cells,neuronal cells and/or liver cells). In certain embodiments, thedisclosure provides a chimeric polypeptide comprising (i) a GAApolypeptide (e.g., a GAA polypeptide comprising or consisting of matureGAA polypeptide), laforin polypeptide, alpha-amylase polypeptide, malinpolypeptide and/or an AGL polypeptide; and (ii) an internalizing moietythat promotes delivery into cells, such as into lysosomes or vacuoles(autophagic vacuoles) of cells (e.g., into lysosomes or vacuoles ofmuscle cells and/or liver cells). Unless specifically indicatedotherwise, delivery into cytoplasm means delivery into, at least,cytoplasm, and GAA, laforin, alpha-amylase, malin and/or AGL activitymay also be delivered to other cellular compartments, such as lysosomesor vacuoles. In a particular embodiment, the internalizing moiety helpsdelivery of the chimeric polypeptide into muscle cells, such as skeletalmuscle cells and/or cardiac muscle cells. In another particularembodiment, the internalizing moiety helps delivery of the chimericpolypeptide into neuronal or liver cells.

In certain embodiments, the disclosure provides chimeric polypeptidesfor delivering GAA, laforin, alpha-amylase, malin and/or AGL activityinto cells, such as into cytoplasm of cells.

Endogenous human GAA is a 952 amino acid protein, encoded by a gene ofapproximately 28 kb in length. In humans, 3 transcript variants areknown (NM_000152.3 which encodes NP000143.2; NM_001079803.1 whichencodes NP_001073271.1; and NM_001079804.1 which encodesNP_001073272.1). However, all three transcript variants encode a proteinhaving substantially the same amino acid sequence. Endogenously, the GAAgene encodes a 952 or 957 amino acid polypeptide which includes a signalsequence. This polypeptide is glycosylated in the endoplasmic reticulumand the Golgi apparatus, resulting in a glycosylated precursor with anapparent molecular mass of 110 kDa. There are 7 potential glycosylationsites on the immature precursor, located at residues 140, 233, 390, 470,652, 882, and 925 of SEQ ID NOs: 1 or 2. The immature precursor istargeted to the lysosomes through mannose-6-phophate receptors (MPRs)and a mannose-6-phosphate (M6P)-independent pathway. The 110 kDaprecursor protein is cleaved to give rise to an endosomal intermediateform of GAA having a molecular weight of about 95 kDa. SubsequentN-terminal and C-terminal proteolytic cleavages generate, in thelysosome, mature, active forms of GAA having molecular weights of about76 kDa and about 70 kDa (Moreland et al., Lysosomal Acid a-GlucosidaseConsists of Four Different Peptides Processed from a Single ChainPrecursor, Journal of Biological Chemistry, 280(8): 6780-6791, 2005;which is incorporated by reference in its entirety). Owing toheterogeneity in the cleavage sites, alternative starting residuesand/or ending residues may define the N and C terminal boundaries ofmature GAA polypeptides, such as mature GAA polypeptides for use in theany of the methods disclosed herein. For example, the N-terminal residueof a mature GAA polypeptide of about 76 kDa may, in certain embodiments,correspond to residue 122 (Met) or 123 (Gly) of SEQ ID NOs: 1 or 2,while the N-terminal residue of a mature GAA polypeptide of about 70 kDamay, in certain embodiments, correspond to any of residues 204 (Ala),206 (Ser), or 288 (Gly) of SEQ ID NOs: 1 or 2. Polypeptides having anyof the foregoing N-terminal residues may have, for example, a C-terminalresidue corresponding to any of residues 816 through 881 of SEQ ID NO:1or 2, and may be residue 782 of SEQ ID NOs: 1 or 2. Additionally, theC-terminal residue may be any of residues 782 through 816, or residues782 through 881, inclusive. The molecular weight of the mature GAApolypeptides may be about 76 kDa or about 70 kDa, or may vary accordingto the foregoing alternative starting and/or ending N and C terminalresidues (e.g., corresponding to portions generated due to alternativecleavage).

The FDA approved a version of GAA referred to as alglucosidase alfa(Myozyme®, Genzyme Corporation), a recombinant human GAA (rhGAA) analogof the 110 kDa precursor form of GAA, produced in CHO cells. Myozyme® isbelieved to be targeted to the endocytic/lysosomal pathway, and isthought to exert its effects in the lysosome. Myozyme® does not appearto treat glycogen accumulation in cytoplasm (Schoser et al., Therapeuticapproaches in Glycogen Storage Disease type II (GSDII)/Pompe Disease,

Neurotherapeutics, 5(4): 569-578, 2008). As noted above, this therapy isbelieved to target the lysosome and is based on delivery of the immatureprecursor form of the protein. However, the precursor form of theprotein is less active than the 76 kDa mature form of the GAA (HumanMolecular Genetics, 7(11): 1815-1824, 1998). Thus, in certain aspects,it may be beneficial to either (i) deliver a mature form of GAA as achimeric polypeptide, (ii) deliver a GAA polypeptide that, althoughlonger than the mature form is shorter than the 110 kDa precursor formas a chimeric polypeptide, and/or (iii) to deliver a GAA polypeptidewith activity of any size as a chimeric polypeptide connected to aninternalizing moiety to facilitate delivery of polypeptide into cells,and even into the appropriate subcellular compartment. Without beingbound by theory, even if a polypeptide of the disclosure hassubstantially the same activity as a precursor GAA polypeptide, deliveryto the proper cellular location, optionally facilitated by aninternalizing moiety that promotes delivery to the cytoplasm, wouldincrease the effective GAA activity delivered to cells. In certainembodiments, the disclosure provides a chimeric polypeptide comprising aGAA polypeptide comprising a full-length GAA polypeptide (e.g., a GAApolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2). Incertain embodiments, the disclosure provides a chimeric polypeptidecomprising a GAA polypeptide of approximately 110 kDa. In certainembodiments, the disclosure provides a chimeric polypeptide comprising aGAA polypeptide comprising mature GAA (a GAA portion comprising matureGAA) and an internalizing moiety portion that facilitate deliver intocells. In other words, the disclosure contemplates chimeric polypeptidescomprising a GAA polypeptide and an internalizing moiety. Numerousexamples of GAA polypeptides suitable for use in the chimericpolypeptides of the disclosure are provided herein. Any such chimericpolypeptide having enzymatic activity is suitable for using in any ofthe methods described herein.

In certain embodiments, the disclosure provides a chimeric polypeptidecomprising a GAA polypeptide and an internalizing moiety, as describedherein. Any such chimeric polypeptide of the disclosure can comprise orconsist of any of the GAA polypeptides described herein associated withany of the internalizing moiety portions described herein, and thesechimeric polypeptides can be used in any of the methods of thedisclosure.

In certain embodiments, chimeric polypeptides of the disclosure comprisea mature GAA polypeptide and may also contain some additional contiguousamino acid sequence from a GAA polypeptide (including the 110 kDprecursor polypeptide or the signal sequence of the GAA precursorpolypeptide). In other embodiments, the chimeric polypeptides of thedisclosure comprise a mature GAA polypeptide but do not includeadditional contiguous amino acid sequence from a GAA polypeptide otherthan the mature GAA polypeptide. Thus, the disclosure contemplateschimeric polypeptides in which the GAA portion comprises or consists ofa mature GAA polypeptide. Exemplary mature GAA polypeptides having amolecular weight of 70-76 kD are described herein. In certainembodiments, the chimeric polypeptide does not include the signalsequence of the precursor GAA polypeptide. In certain embodiments, thechimeric polypeptide does not include a portion corresponding toresidues 1-56 of SEQ ID NO: 1 or 2 and/or a portion corresponding toresidues 1-57 of SEQ ID NO: 1 or 2 (e.g., the GAA polypeptide does notinclude a portion corresponding to residues 1-56 and/or residues 1-57 ofSEQ ID NO: 1 or 2). In other embodiments, the chimeric polypeptidecomprises the entire immature GAA polypeptide (e.g., the amino acidsequences of either SEQ ID NOs: 1 or 2). It is noted that a GAApolypeptide comprising mature GAA is also referred to as a GAApolypeptide comprising mature GAA polypeptide. Such a GAA polypeptide,and any of the GAA polypeptides provided herein, may be a singlepolypeptide chain.

In certain embodiments, the GAA polypeptide portion comprises the aminoacid sequence of SEQ ID NO: 21 (e.g., the GAA polypeptide comprises SEQID NO: 21), and thus, the chimeric polypeptide comprises a mature GAAhaving the amino acid sequence of SEQ ID NO: 3 or 4. In certainembodiments, the chimeric polypeptide does not include additionalcontiguous amino acid sequence from human GAA—other than SEQ ID NO: 21.In certain embodiments, the GAA polypeptide or chimeric polypeptide doesnot include residues 1-56 of SEQ ID NO: 1. In certain embodiments, theGAA polypeptide or chimeric polypeptide does not include residues 1-60of SEQ ID NO: 1. In certain embodiments, the GAA polypeptide portioncomprises the amino acid sequence of SEQ ID NO: 22 (e.g., the GAApolypeptide comprises SEQ ID NO: 22), and thus, the chimeric polypeptidecomprises a mature GAA having the amino acid sequence of SEQ ID NO: 3 or4. In certain embodiments, the chimeric polypeptide does not includeadditional contiguous amino acid sequence from human GAA—other than SEQID NO: 22. In certain embodiments, the AA polypeptide or chimericpolypeptide does not include residues 1-66 of SEQ ID NO: 1. In certainembodiments, the GAA polypeptide portion comprises the amino acidsequence of SEQ ID NO: 23 (e.g., the GAA polypeptide comprises SEQ IDNO: 23), and thus, the chimeric polypeptide comprises a mature GAAhaving the amino acid sequence of SEQ ID NO: 3 or 4. In certainembodiments, the chimeric polypeptide does not include additionalcontiguous amino acid sequence from human GAA—other than SEQ ID NO: 23.In certain embodiments, the GAA polypeptide or chimeric polypeptide doesnot include residues 1-69 of SEQ ID NO: 1.

As used herein, the GAA polypeptides include variants, and, in someembodiments, the mature, active forms of the protein (the active about76 kDa or about 70 kDa forms or similar forms having an alternativestarting and/or ending residue, collectively termed “mature GAA”). Theterm “mature GAA” refers to a polypeptide having an amino acid sequencecorresponding to that portion of the immature GAA protein that, whenprocessed endogenously, has an apparent molecular weight by SDS-PAGE ofabout 70 kDa to about 76 kDa, as well as similar polypeptides havingalternative starting and/or ending residues, as described above. In someembodiments, the GAA polypeptide lacks the signal sequence (amino acids1-27 of SEQ ID NOs: 1 or 2 or the sequence designated by amino acids1-56 of SEQ ID NO: 1 or 2). Exemplary mature GAA polypeptides includepolypeptides having residues 122-782 of SEQ ID NOs: 1 or 2; residues123-782 of SEQ ID NOs: 1 or 2; or residues 204-782 of SEQ ID NOs: 1 or2.

The term “GAA” includes polypeptides (e.g., mature GAA polypeptides)that are glycosylated in the same or substantially the same way as theendogenous, mature proteins, and thus have a molecular weight that isthe same or similar to the predicted molecular weight. The term alsoincludes polypeptides that are not glycosylated or arehyper-glycosylated, such that their apparent molecular weight differdespite including the same primary amino acid sequence. Any suchvariants or isoforms, functional fragments or variants, fusion proteins,and modified forms of the GAA polypeptides have at least a portion ofthe amino acid sequence of substantial sequence identity to the nativeGAA protein, and retain enzymatic activity. In certain embodiments, afunctional fragment, variant, or fusion protein of a mature GAApolypeptide comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to mature GAA polypeptides setforth in one or both of SEQ ID NOs: 3 and 4, or is at least 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to mature GAA polypeptidescorresponding to one or more of: residues 122-782 of SEQ ID NOs: 1 or 2;residues 123-782 of SEQ ID NOs: 1 or 2; or residues 204-782 of SEQ IDNOs: 1 or 2. In some embodiments, the GAA polypeptide is a GAApolypeptide from a non-human species, e.g., mouse, rat, dog, zebrafish,pig, goat, cow, horse, monkey or ape. In some embodiments, the GAAprotein comprises a bovine GAA protein or fragment thereof (e.g., themature form) having the amino acid sequence of SEQ ID NO: 32.

In certain embodiments, the GAA polypeptide portion (e.g., the GAApolypeptide) of any of the chimeric polypeptides disclosed hereincomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1 or 2. In certain embodiments, the GAA polypeptide portion of any ofthe chimeric polypeptides disclosed herein comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to a sequence corresponding to residues 57-782 of the aminoacid sequence of SEQ ID NO: 1 or 2. In certain embodiments, the GAApolypeptide portion of any of the chimeric polypeptides disclosed hereincomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to a sequence corresponding to residues67-782 of the amino acid sequence of SEQ ID NO: 1 or 2. In certainembodiments, the GAA polypeptide portion (e.g., the GAA polypeptide) ofany of the chimeric polypeptides disclosed herein comprises an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of a sequence corresponding toresidues 57-952 of SEQ ID NO: 1 or 2. In certain embodiments, the GAApolypeptide portion of any of the chimeric polypeptides disclosed hereincomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to a sequence corresponding to residues67-952 of the amino acid sequence of SEQ ID NO: 1 or 2. In certainembodiments, the GAA polypeptide comprises an amino acid sequence thatis at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to theamino acid sequence set forth in SEQ ID NO: 21, 22, and/or 23.

In certain embodiments, a GAA polypeptide for use in a chimericpolypeptide comprises 1, 2, 3, 4, or 5 amino acid substitutions,relative to the corresponding portion of human GAA set forth in SEQ IDNO: 1 or 2. In certain embodiments, a GAA polypeptide for use in achimeric polypeptide comprises 1, 2, 3, 4, or 5 amino acidsubstitutions, relative to the human GAA polypeptide set forth in SEQ IDNO: 21, 22 and/or 23 (e.g., the GAA polypeptide comprises or consists ofSEQ ID NO: 21, 22 and/or 23, but with 1, 2, 3, 4, or 5 amino acidsubstitutions, relative to SEQ ID NO: 21, 22 and/or 23). In certainembodiments, a GAA polypeptide for use in a chimeric polypeptidecomprises or consists of SEQ ID NO: 21, 22 and/or 23, but differs by 1,2, 3, 4, or 5 amino acid residues at its N- or C-terminus, such as has1, 2, 3, 4 or 5 amino acid residues deleted at the N and/or C-terminus.

GAA polypeptides having any combination of the structural and functionalcharacteristics described herein are specifically contemplated.

Here and elsewhere in the specification, sequence identity refers to thepercentage of residues in the candidate sequence that are identical withthe residue of a corresponding sequence to which it is compared, afteraligning the sequences and introducing gaps, if necessary to achieve themaximum percent identity for the entire sequence, and not consideringany conservative substitutions as part of the sequence identity. Incertain embodiments, neither N- or C-terminal extensions nor insertionsshall be construed as reducing identity or homology.

Methods and computer programs for the alignment of sequences and thecalculation of percent identity are well known in the art and readilyavailable. Sequence identity may be measured using sequence analysissoftware. For example, alignment and analysis tools available throughthe ExPasy bioinformatics resource portal, such as ClustalW algorithm,set to default parameters. Suitable sequence alignments and comparisonsbased on pair-wise or global alignment can be readily selected. Oneexample of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J Mol Biol 215:403-410 (1990). Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (www.ncbi.nlm.nih.gov/). In certainembodiments, the now current default settings for a particular programare used for aligning sequences and calculating percent identity.

In certain specific embodiments, the chimeric polypeptide comprises aGAA polypeptide, such as a GAA polypeptide comprising mature GAA. TheGAA has an activity that is similar to or substantially equivalent tothe activity of endogenous forms of human GAA (e.g., the 110 kDaprecursor form of GAA). In certain embodiments, the mature GAA has anactivity that is similar to or substantially equivalent to the activityof endogenous forms of human GAA that are about 76 kDa or about 70 kDa.For example, the mature GAA may be 7-10 fold more active for glycogenhydrolysis than the 110 kDa precursor form, with the comparison beingmade under the same or similar conditions (e.g. the mature GAA-chimericpolypeptides disclosed herein as compared with endogenous human immatureprecursor GAA under acidic or neutral pH conditions). The mature GAApolypeptide may be the 76 kDa or the 70 kDa form of GAA, or similarforms that use alternative starting and/or ending residues. As noted inMoreland et al. (Lysosomal Acid α-Glucosidase Consists of Four DifferentPeptides Processed from a Single Chain Precursor, Journal of BiologicalChemistry, 280(8): 6780-6791, 2005), the nomenclature used for theprocessed forms of GAA is based on an apparent molecular mass asdetermined by SDS-PAGE. In some embodiments, mature GAA may lack theN-terminal sites that are normally glycosylated in the endoplasmicreticulum. An exemplary mature GAA polypeptide comprises SEQ ID NO: 3 orSEQ ID NO: 4. Further exemplary mature GAA polypeptide may comprise orconsist of an amino acid sequence corresponding to about: residues122-782 of SEQ ID NOs: 1 or 2; residues 123-782 of SEQ ID NOs: 1 or 2,such as shown in SEQ ID NO: 3; residues 204-782 of SEQ ID NOs: 1 or 2;residues 206-782 of SEQ ID NOs: 1 or 2; residues 288-782 of SEQ ID NOs:1 or 2, as shown in SEQ ID NO: 4. Mature GAA polypeptides may also havethe N-terminal and or C-terminal residues described above.

In certain embodiments, the chimeric polypeptide comprises a full-lengthGAA polypeptide, e.g., the chimeric polypeptide comprises the amino acidsequences of SEQ ID NOs: 1 or 2. In certain embodiments, the chimericpolypeptide does not comprise a full-length GAA polypeptide, butcomprises a mature GAA polypeptide and at least a portion of thefull-length GAA polypeptide. In other words, in certain embodiments, thechimeric polypeptide comprises a GAA polypeptide and an internalizingmoiety. In some embodiments, the chimeric polypeptide does not comprisea full-length GAA polypeptide comprising the amino acid sequence of SEQID NO: 1 or 2, but comprises a mature GAA polypeptide sequencecomprising the amino acid sequences of SEQ ID NOs: 3 or 4 and at least aportion of the amino acids corresponding to amino acids 1-121 of SEQ IDNOs: 1-2 (e.g., a portion of contiguous amino acids) and/or at least aportion of the amino acids corresponding to amino acids 783-952 of SEQID NO: 1 (e.g., a portion of contiguous amino acids). In someembodiments, the chimeric polypeptide does not comprise a full-lengthGAA polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2,but comprises a mature GAA polypeptide sequence comprising the aminoacid sequences of SEQ ID NOs: 3 or 4 and at least a portion of the aminoacids corresponding to amino acids 783-952 of SEQ ID NO: 1 (e.g., aportion of contiguous amino acids). In some embodiments, the chimericpolypeptide does not comprise a full-length GAA polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1 or 2, but comprises a mature GAApolypeptide sequence comprising the amino acid sequences of SEQ ID NOs:3 or 4 and at least a portion of the amino acids corresponding to aminoacids 783-957 of SEQ ID NO: 2 (e.g., a portion of contiguous aminoacids). These are exemplary of GAA polypeptides.

In certain embodiments, the GAA polypeptide portion (e.g., the portioncomprising a GAA polypeptide comprising mature GAA; e.g., a GAApolypeptide) of the chimeric proteins described herein comprise a matureform of GAA but does not comprise a GAA translation product set forth inSEQ ID NO: 1. In some embodiments, neither the GAA polypeptide nor thechimeric polypeptide comprise a contiguous amino acid sequencecorresponding to the amino acids 1-27 or 1-56 of SEQ ID NO: 1 or 2. Insome embodiments, the GAA polypeptide lacks at least a portion of theGAA full linker region, wherein the full linker region corresponds toamino acids 57-78 of SEQ ID NOs: 1 or 2 (i.e., SEQ ID NO: 31). In someembodiments, the GAA polypeptide does not comprise a contiguous aminoacid sequence corresponding to any one or more of the amino acids 1-27,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65,1-66, 1-67, 1-68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-80, 1-85,1-90, 1-95, 1-100, 1-105, 1-110, 1-115, 1-120 or 1-121 of SEQ ID NOs: 1or 2. In other embodiments, the GAA polypeptide does comprise any one ormore of the foregoing.

In particular embodiments, the GAA polypeptide does not comprise acontiguous amino acid sequence corresponding to the amino acids 1-60 ofSEQ ID NOs: 1 or 2 (e.g., the chimeric polypeptide comprises a GAApolypeptide portion comprising a GAA polypeptide comprising the aminoacid sequence of SEQ ID NO: 21). In other embodiments, the GAA portiondoes not comprise a contiguous amino acid sequence corresponding to theamino acids 1-66 of SEQ ID NO: 1 or 2 (e.g., the chimeric polypeptidecomprises a GAA polypeptide comprising the amino acid sequence of SEQ IDNO: 22). In some embodiments, the GAA portion does not comprise acontiguous amino acid sequence corresponding to the amino acids 1-69 ofSEQ ID NO: 1 or 2 (e.g., the chimeric polypeptide comprises a GAApolypeptide portion comprising a GAA polypeptide comprising the aminoacid sequence of SEQ ID NO: 23).

In some embodiments, the GAA polypeptides may be glycosylated, or may benot glycosylated. For those GAA polypeptides that are glycosylated, theglycosylation pattern may be the same as that of naturally-occurringhuman GAA or may be different. In some embodiments, one or more of theglycosylation sites on the precursor GAA protein may be removed in thefinal mature GAA construct.

GAA has been isolated from tissues such as bovine testes, rat liver, pigliver, human liver, rabbit muscle, human heart, human urine, and humanplacenta. GAA (e.g., GAA) may also be produced using recombinanttechniques, for example by transfecting Chinese hamster ovary (CHO)cells with a vector that expresses full-length human GAA or a vectorthat expresses mature GAA. Recombinant human GAA (rhGAA) or mature GAAis then purified from CHO-conditioned medium, using a series ofultrafiltration, diafiltration, washing, and eluting steps, as describedby Moreland et al. (Lysosomal Acid a-Glucosidase Consists of FourDifferent Peptides Processed from a Single Chain Precursor, Journal ofBiological Chemistry, 280(8): 6780-6791, 2005). GAA fragments may beseparated according to methods known in the art, such as affinitychromatography and SDS page.

In certain embodiments, GAA (e.g., mature GAA), or fragments or variantsare human GAA.

In certain embodiments, fragments or variants of the GAA polypeptidescan be obtained by screening polypeptides recombinantly produced fromthe corresponding fragment of the nucleic acid encoding a GAApolypeptide. In addition, fragments or variants can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. The fragments orvariants can be produced (recombinantly or by chemical synthesis) andtested to identify those fragments or variants that can function as anative GAA protein, for example, by testing their ability hydrolyzeglycogen and/or treat symptoms of Forbes-Cori and/or Andersen Diseaseand/or Pompe Disease and/or von Gierke's Disease and/or Lafora Disease.

In certain embodiments, the present disclosure contemplates modifyingthe structure of a GAA polypeptide (e.g., mature GAA polypeptide) forsuch purposes as enhancing therapeutic or prophylactic efficacy, orstability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo). Such modified GAA polypeptides are consideredfunctional equivalents of the naturally-occurring GAA polypeptide.Modified polypeptides can be produced, for instance, by amino acidsubstitution, deletion, or addition. For instance, it is reasonable toexpect, for example, that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the GAA biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains.

This disclosure further contemplates generating sets of combinatorialmutants of an GAA polypeptide (e.g., mature GAA polypeptide), as well astruncation mutants, and is especially useful for identifying functionalvariant sequences. Combinatorially-derived variants can be generatedwhich have a selective potency relative to a naturally occurring GAApolypeptide. Likewise, mutagenesis can give rise to variants which haveintracellular half-lives dramatically different than the correspondingwild-type GAA polypeptide. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular process which result in destruction of, or otherwiseinactivation of GAA function. Such variants can be utilized to alter theGAA polypeptide level by modulating their half-life. There are many waysby which the library of potential GAA variants sequences can begenerated, for example, from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then be ligatedinto an appropriate gene for expression. The purpose of a degenerate setof genes is to provide, in one mixture, all of the sequences encodingthe desired set of potential polypeptide sequences. The synthesis ofdegenerate oligonucleotides is well known in the art (see for example,Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) RecombinantDNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton,Amsterdam: Elsevier pp273-289; Itakura et al., (1984) Annu. Rev.Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al.,(1983) Nucleic Acid Res. 11:477). Such techniques have been employed inthe directed evolution of other proteins (see, for example, Scott etal., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al.,(1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, GAA polypeptide (e.g., mature GAApolypeptide) variants can be generated and isolated from a library byscreening using, for example, alanine scanning mutagenesis and the like(Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J.Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118;Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al.,(1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085), bylinker scanning mutagenesis (Gustin et al., (1993) Virology 193:653-660;Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al.,(1982) Science 232:316); by saturation mutagenesis (Meyers et al.,(1986) Science 232:613); by PCR mutagenesis (Leung et al., (1989) MethodCell Mol Biol 1:11-19); or by random mutagenesis, including chemicalmutagenesis, etc. (Miller et al., (1992) A Short Course in BacterialGenetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greener et al.,(1994) Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis,particularly in a combinatorial setting, is an attractive method foridentifying truncated (bioactive) forms of GAA.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of the GAA polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

In certain embodiments, a GAA polypeptide may include a peptide and apeptidomimetic. As used herein, the term “peptidomimetic” includeschemically modified peptides and peptide-like molecules that containnon-naturally occurring amino acids, peptoids, and the like.Peptidomimetics provide various advantages over a peptide, includingenhanced stability when administered to a subject. Methods foridentifying a peptidomimetic are well known in the art and include thescreening of databases that contain libraries of potentialpeptidomimetics. For example, the Cambridge Structural Database containsa collection of greater than 300,000 compounds that have known crystalstructures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)).Where no crystal structure of a target molecule is available, astructure can be generated using, for example, the program CONCORD(Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Anotherdatabase, the Available Chemicals Directory (Molecular Design Limited,Informations Systems; San Leandro Calif.), contains about 100,000compounds that are commercially available and also can be searched toidentify potential peptidomimetics of the mature GAA polypeptides.

In certain embodiments, a GAA polypeptide may further comprisepost-translational modifications. Exemplary post-translational proteinmodification include phosphorylation, acetylation, methylation,ADP-ribosylation, ubiquitination, glycosylation, carbonylation,sumoylation, biotinylation or addition of a polypeptide side chain or ofa hydrophobic group. As a result, the modified GAA polypeptides maycontain non-amino acid elements, such as lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of a GAA polypeptide may be tested for itsbiological activity, for example, its ability to treat Forbes-Coriand/or Andersen Disease and/or Pompe Disease and/or von Gierke Diseaseand/or Lafora Disease, and/or its ability to decrease glycogenaccumulation in cytoplasm and/or lysosomes of Forbes-Cori Disease and/orAndersen Disease and/or von Gierke Disease and/or Pompe Disease and/orLafora Disease cells. Biological activity of GAA may also be evaluatedin a cell free or cell-based enzymatic assay. In certain embodiments,the GAA polypeptide may further comprise one or more polypeptideportions that enhance one or more of in vivo stability, in vivo halflife, uptake/administration, and/or purification. In other embodiments,the internalizing moiety comprises an antibody or an antigen-bindingfragment thereof.

In one specific embodiment of the present disclosure, a GAA polypeptidemay be modified with nonproteinaceous polymers. In one specificembodiment, the polymer is polyethylene glycol (“PEG”), polypropyleneglycol, or polyoxyalkylenes, in the manner as set forth in U.S. Pat.Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161).

By the terms “biological activity”, “bioactivity” or “functional” ismeant the ability of the GAA protein to carry out the functionsassociated with wildtype GAA proteins, for example, the hydrolysis ofa-1,4- and a-1,6-glycosidic linkages of glycogen, for example lysosomalglycogen or cytoplasmic glycogen. The terms “biological activity”,“bioactivity”, and “functional” are used interchangeably herein. Incertain embodiments, and as described herein, a GAA protein or chimericpolypeptide having biological activity has the ability to hydrolyzeglycogen. In other embodiments, a GAA protein or chimeric polypeptidehaving biological activity has the ability to lower the concentration oflysosomal, vacuolar (e.g. autophagic vacuolar) and/or cytoplasmicglycogen. In still other embodiments, a GAA protein or chimericpolypeptide has the ability to treat symptoms associated with PompeDisease and/or Forbes-Cori and/or Andersen Disease and/or von GierkeDisease and/or Lafora Disease. As used herein, “fragments” areunderstood to include bioactive fragments (also referred to asfunctional fragments) or bioactive variants that exhibit “bioactivity”as described herein. That is, bioactive fragments or variants of GAAexhibit bioactivity that can be measured and tested. For example,bioactive fragments/functional fragments or variants exhibit the same orsubstantially the same bioactivity as native (i.e., wild-type, ornormal) GAA protein, and such bioactivity can be assessed by the abilityof the fragment or variant to, e.g., hydrolyze glycogen in vitro or invivo. As used herein, “substantially the same” refers to any parameter(e.g., activity) that is at least 70% of a control against which theparameter is measured. In certain embodiments, “substantially the same”also refers to any parameter (e.g., activity) that is at least 75%, 80%,85%, 90%, 92%, 95%, 97%, 98%, 99%, 100%, 102%, 105%, or 110% of acontrol against which the parameter is measured, when assessed under thesame or substantially the same conditions. In certain embodiments,fragments or variants of the GAA polypeptide will preferably retain atleast 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of the GAA biologicalactivity associated with the native GAA polypeptide, when assessed underthe same or substantially the same conditions. In certain embodiments,fragments or variants of the GAA polypeptide have a half-life (t_(1/2))which is enhanced relative to the half-life of the native protein.Preferably, the half-life of GAA fragments or variants is enhanced by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%,175%, 200%, 250%, 300%, 400% or 500%, or even by 1000% relative to thehalf-life of the native GAA protein, when assessed under the same orsubstantially the same conditions. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf life, such as the half-life of the protein in the serum or otherbodily fluid of an animal. In addition, fragments or variants can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments or variants can be produced (recombinantly or by chemicalsynthesis) and tested to identify those fragments or variants that canfunction as well as or substantially similarly to a native GAA protein.

With respect to methods of increasing GAA bioactivity in cells, thedisclosure contemplates all combinations of any of the foregoing aspectsand embodiments, as well as combinations with any of the embodiments setforth in the detailed description and examples. The described methodsbased on administering chimeric polypeptides or contacting cells withchimeric polypeptides can be performed in vitro (e.g., in cells orculture) or in vivo (e.g., in a patient or animal model). In certainembodiments, the method is an in vitro method. In certain embodiments,the method is an in vivo method. In certain embodiments, administering achimeric polypeptide of the disclosure increases GAA bioactivity incells, in vitro and/or in vivo, and methods of doing so are provided.

In some aspects, the present disclosure also provides a method ofproducing any of the foregoing chimeric polypeptides as describedherein. Further, the present disclosure contemplates any number ofcombinations of the foregoing methods and compositions.

In certain aspects, a GAA polypeptide (e.g., mature GAA polypeptide) maybe a fusion protein which further comprises one or more fusion domains.Well-known examples of such fusion domains include, but are not limitedto, polyhistidine, Glu-Glu, glutathione S transferase (GST),thioredoxin, protein A, protein G, and an immunoglobulin heavy chainconstant region (Fc), maltose binding protein (MBP), which areparticularly useful for isolation of the fusion proteins by affinitychromatography. For the purpose of affinity purification, relevantmatrices for affinity chromatography, such as glutathione-, amylase-,and nickel- or cobalt- conjugated resins are used. Fusion domains alsoinclude “epitope tags,” which are usually short peptide sequences forwhich a specific antibody is available. Well known epitope tags forwhich specific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), His, and c-myc tags. An exemplaryHis tag has the sequence HHHHHH (SEQ ID NO: 7), and an exemplary c-myctag has the sequence EQKLISEEDL (SEQ ID NO: 8). It is recognized thatany such tags or fusions may be appended to the GAA portion of thechimeric polypeptide or may be appended to the internalizing moietyportion of the chimeric polypeptide, or both. In certain embodiments,the chimeric polypeptides comprise a “AGIH” portion (SEQ ID NO: 19) onthe N-terminus (or within 10 amino acid residues of the N-terminus) ofthe chimeric polypeptide, and such chimeric polypeptides may be providedin the presence or absence of one or more epitope tags. In furtherembodiments, the chimeric polypeptide comprises a serine at theN-terminal most position of the polypeptide. In some embodiments, thechimeric polypeptides comprise an “SAGIH” (SEQ ID NO: 20) portion at theN-terminus (or within 10 amino acid residues of the N-terminus) of thepolypeptide, and such chimeric polypeptides may be provided in thepresence or absence of one or more epitope tags.

In some cases, the fusion domains have a protease cleavage site, such asfor Factor Xa or Thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. In certain embodiments, the GAA polypeptides may contain oneor more modifications that are capable of stabilizing the polypeptides.For example, such modifications enhance the in vitro half life of thepolypeptides, enhance circulatory half life of the polypeptides orreducing proteolytic degradation of the polypeptides.

In certain embodiments of any of the foregoing, the GAA portion of thechimeric protein comprises one of the mature forms of GAA, e.g., the 76kDa fragment, the 70 kDa fragment, similar forms that use an alternativestart and/or stop site, or a functional fragment thereof. In certainembodiments, such mature GAA polypeptide or functional fragment thereofretains the ability of to hydrolyze glycogen, as evaluated in vitro orin vivo. Further, in certain embodiments, the chimeric polypeptide thatcomprises such a mature GAA polypeptide or functional fragment thereofcan hydrolyze glycogen. Exemplary bioactive fragments comprise at least50, at least 60, at least 75, at least 100, at least 125, at least 150,at least 175, at least 200, at least 225, at least 230, at least 250, atleast 260, at least 275, or at least 300 consecutive amino acid residuesof a full length mature GAA polypeptide.

In certain embodiments, the GAA polypeptide portion of the chimericpolypeptides described herein comprise a full-length immature form ofGAA (e.g., a polypeptide comprising the amino acid sequence of SEQ IDNOs: 1 and 2, in the presence or absence of the signal sequence). Incertain embodiments, the GAA polypeptide portion of the chimericproteins described herein comprise a GAA (e.g., consecutive GAApolypepetide sequence that comprises mature GAA) but does not comprise aGAA polypeptide set forth in SEQ ID NO: 1. In some embodiments, the GAApolypeptide lacks at least a portion of the GAA full linker region,wherein the full linker region corresponds to amino acids 57-78 of SEQID NOs: 1 or 2 (i.e., SEQ ID NO: 31). In some embodiments, the GAApolypeptide does not comprise a contiguous amino acid sequencecorresponding to any one or more of the following: the amino acids 1-27,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65,1-66, 1-67, 1-68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-80, 1-85,1-90, 1-95, 1-100, 1-105, 1-110, 1-115, 1-120 or 1-121 of SEQ ID NOs: 1or 2. In other words, in certain embodiments, the chimeric polypeptidelacks any one of the foregoing. In other embodiments, the GAApolypeptide does comprise any one or more of the foregoing. Inparticular embodiments, the GAA polypeptide does not comprise acontiguous amino acid sequence corresponding to the amino acids 1-60 ofSEQ ID NOs: 1 or 2 (e.g., the chimeric polypeptide comprises a GAApolypeptide comprising the amino acid sequence of SEQ ID NO: 21 and, incertain embodiments, the chimeric polypeptide does not comprise aminoacids 1-60 of SEQ ID NO: 1 or 2). In other embodiments, the GAA portiondoes not comprise a contiguous amino acid sequence corresponding to theamino acids 1-66 of SEQ ID NO: 1 or 2 (e.g., the chimeric polypeptidecomprises a GAA polypeptide comprising the amino acid sequence of SEQ IDNO: 22 and, in certain embodiments, the chimeric polypeptide does notcomprise a contiguous amino acid sequence corresponding to amino acids1-60 or 1-66 of SEQ ID NO: 1 or 2). In some embodiments, the GAA portiondoes not comprise a contiguous amino acid sequence corresponding to theamino acids 1-69 of SEQ ID NO: 1 or 2 (e.g., the chimeric polypeptidecomprises a GAA polypeptide portion comprising a GAA polypeptidecomprising the amino acid sequence of SEQ ID NO: 23 and, in certainembodiments, the chimeric polypeptide does not comprise a contiguousamino acid sequence corresponding to amino acids 1-60 or 1-66 or 1-69 ofSEQ ID NO: 1 or 2). Suitable combinations, as set forth herein, arespecifically contemplated. Chimeric polypeptides comprising any such GAApolypeptides comprising mature GAA may be used to deliver GAA activityinto cells.

In certain embodiments, the disclosure contemplates chimeric proteinswhere the GAA portion (e.g., a mature GAA portion) is a variant of anyof the foregoing GAA polypeptides or functional fragments. Exemplaryvariants have an amino acid sequence at least 90%, 92%, 95%, 96%, 97%,98%, or at least 99% identical to the amino acid sequence of a nativeGAA polypeptide or bioactive fragment thereof, and such variants retainthe ability of native GAA to hydrolyze glycogen, as evaluated in vitroor in vivo. The disclosure contemplates chimeric proteins and the use ofsuch proteins wherein the GAA portion comprises any of the GAApolypeptides (e.g., mature GAA polypeptides), forms, or variantsdescribed herein in combination with any internalizing moiety describedherein. Exemplary mature GAA polypeptides are set forth in SEQ ID NOs: 3and 4. Exemplary GAA polypeptides comprising mature GAA are set forthherein. Moreover, in certain embodiments, the GAA portion of any of theforegoing chimeric polypeptides may, in certain embodiments, by a fusionprotein. Any such chimeric polypeptides comprising any combination ofGAA portions and internalizing moiety portions, and optionally includingone or more linkers, one or more tags, etc., may be used in any of themethods of the disclosure.

In certain embodiments, the disclosure contemplates chimeric polypeptidecomprising a GAA polypeptide, as described herein. In certainembodiments, chimeric polypeptides of the disclosure comprise a GAApolypeptide portion (e.g., the non-internalizing moiety polypeptideportion comprises a GAA polypeptide). Suitable chimeric polypeptides ofthe disclosure have enzymatic activity and may be used to decreaseglycogen accumulation in cytoplasm, such as in subjects having GSD III,GSD IV, Pompe Disease, and/or GSD I (including GSD1a or GSD1b). Unlessspecifically indicated otherwise, reference to the activity of achimeric polypeptide of the disclosure in cytoplasm refers to havingactivity in, at least, cytoplasm. In certain embodiments, suitablechimeric polypeptides of the disclosure have enzymatic activity and maybe used to decrease glycogen accumulation in lysosomes and vacuoles(e.g., autophagic vacuoles), such as in subjects having GSD III, GSD IV,Pompe Disease, Lafora Disease, or GSD I (including GSD Ia or GSD Ib).

II. Laforin Polypeptides

In certain embodiments, the non-internalizing moiety polypeptide portionof a chimeric polypeptide of the disclosure (or a chimeric polypeptidefor use in the methods of the disclosure) is a laforin polypeptide. Inother words, in certain embodiments, laforin-containing chimericpolypeptides are provided. Exemplary laforin polypeptides for use in themethods and compositions of the disclosure are provided herein.

As used herein, the laforin polypeptides include various functionalfragments and variants, fusion proteins, and modified forms of thewildtype laforin polypeptide. Such functional fragments or variants,fusion proteins, and modified forms of the laforin polypeptides have atleast a portion of the amino acid sequence of substantial sequenceidentity to the native laforin polypeptide, and retain the function ofthe native laforin polypeptide (e.g., protein phosphatase activity,glucan phosphatase activity, the ability to form a complex with malin,and/or glycogen binding activity of native laforin). It should be notedthat “retain the function” does not mean that the activity of aparticular fragment must be identical or substantially identical to thatof the native protein although, in some embodiments, it may be. However,to retain the native activity, that native activity should be at least50%, at least 60%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95% that of the native protein to which suchactivity is being compared, with the comparison being made under thesame or similar conditions. In some embodiments, retaining the nativeactivity may include scenarios in which a fragment or variant hasimproved activity versus the native protein to which such activity isbeing compared, e.g., at least 105%, at least 110%, at least 120%, or atleast 125%, with the comparison being bade under the same or similarconditions.

In certain embodiments, a functional fragment, variant, or fusionprotein of a laforin polypeptide comprises an amino acid sequence thatis at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to alaforin polypeptide (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to SEQ ID NO: 38 or 39), or fragments thereof.

In certain embodiments, the laforin polypeptide for use in the chimericpolypeptides and methods of the disclosure is a full length orsubstantially full length laforin polypeptide. In certain embodiments,the laforin polypeptide for use in the chimeric polypeptide and methodsof the disclosure is a functional fragment that has protein phosphataseactivity, glucan phosphatase activity, the ability to form a complexwith malin, and/or carbohydrate binding activity (e.g., glycogen bindingactivity).

In certain embodiments, fragments or variants of the laforinpolypeptides can be obtained by screening polypeptides recombinantlyproduced from the corresponding fragment of the nucleic acid encoding alaforin polypeptide. In addition, fragments or variants can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments or variants can be produced (recombinantly or by chemicalsynthesis) and tested to identify those fragments or variants that canfunction as a native laforin polypeptide, for example, by testing theirability to treat Lafora Disease in vivo and/or by confirming in vitro(e.g., in a cell free or cell based assay) that the fragment or varianthas protein phosphatase activity, glucan phosphatase activity, abilityto form a complex with malin, and/or carbohydrate binding activity(e.g., glycogen binding activity). An example of an in vitro assay fortesting for activity of the laforin polypeptides disclosed herein wouldbe to treat Lafora cells with or without the laforin-containing chimericpolypeptides and then, after a period of incubation, determining LC3staining in the treated cells as compared to the untreated controlcells. An increase in the amount of LC3 staining in the treated cells ascompared to the untreated control cells is indicative that animprovement in autophagic function may be occurring in the treatedcells.

In certain embodiments, the present disclosure contemplates modifyingthe structure of a laforin polypeptide for such purposes as enhancingtherapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelflife and resistance to proteolytic degradation in vivo). Modifiedpolypeptides can be produced, for instance, by amino acid substitution,deletion, or addition. For instance, it is reasonable to expect, forexample, that an isolated replacement of a leucine with an isoleucine orvaline, an aspartate with a glutamate, a threonine with a serine, or asimilar replacement of an amino acid with a structurally related aminoacid (e.g., conservative mutations) will not have a major effect on thelaforin biological activity of the resulting molecule. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains.

This disclosure further contemplates generating sets of combinatorialmutants of a laforin polypeptide, as well as truncation mutants, and isespecially useful for identifying functional variant sequences.Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring laforin polypeptide. Likewise,mutagenesis can give rise to variants which have intracellularhalf-lives dramatically different than the corresponding wild-typelaforin polypeptide. For example, the altered protein can be renderedeither more stable or less stable to proteolytic degradation or othercellular process which result in destruction of, or otherwiseinactivation of laforin. Such variants can be utilized to alter thelaforin polypeptide level by modulating their half-life. There are manyways by which the library of potential laforin variants sequences can begenerated, for example, from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then can beligated into an appropriate gene for expression. The purpose of adegenerate set of genes is to provide, in one mixture, all of thesequences encoding the desired set of potential polypeptide sequences.The synthesis of degenerate oligonucleotides is well known in the art(see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al.,(1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed.AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al., (1984) Annu.Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike etal., (1983) Nucleic Acid Res. 11:477). Such techniques have beenemployed in the directed evolution of other proteins (see, for example,Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNASUSA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla etal., (1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, laforin polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of the laforin polypeptide.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of the laforin polypeptides. The most widelyused techniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

In certain embodiments, a laforin polypeptide may include apeptidomimetic. As used herein, the term “peptidomimetic” includeschemically modified peptides and peptide-like molecules that containnon-naturally occurring amino acids, peptoids, and the like.Peptidomimetics provide various advantages over a peptide, includingenhanced stability when administered to a subject. Methods foridentifying a peptidomimetic are well known in the art and include thescreening of databases that contain libraries of potentialpeptidomimetics. For example, the Cambridge Structural Database containsa collection of greater than 300,000 compounds that have known crystalstructures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)).Where no crystal structure of a target molecule is available, astructure can be generated using, for example, the program CONCORD(Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Anotherdatabase, the Available Chemicals Directory (Molecular Design Limited,Informations Systems; San Leandro Calif.), contains about 100,000compounds that are commercially available and also can be searched toidentify potential peptidomimetics of the laforin polypeptides.

In certain embodiments, a laforin polypeptide may further comprisepost-translational modifications. Exemplary post-translational proteinmodifications include phosphorylation, acetylation, methylation,ADP-ribosylation, ubiquitination, glycosylation, carbonylation,sumoylation, biotinylation or addition of a polypeptide side chain or ofa hydrophobic group. As a result, the modified laforin polypeptides maycontain non-amino acid elements, such as lipids, poly- ormono-saccharides, and phosphates. Effects of such non-amino acidelements on the functionality of a laforin polypeptide may be tested forits biological activity, for example, its protein phosphatase activity,glucan phosphatase activity, ability to form a complex with malin,and/or carbohydrate binding activity (e.g., glycogen binding activity)and/or its ability to treat Lafora Disease. In certain embodiments, thelaforin polypeptide may further comprise one or more polypeptideportions that enhance one or more of in vivo stability, in vivo halflife, uptake/administration, and/or purification. In other embodiments,the internalizing moiety comprises an antibody or an antigen-bindingfragment thereof.

In some embodiments, a laforin polypeptide is not N-glycosylated orlacks one or more of the N-glycosylation groups present in a wildtypelaforin polypeptide. For example, the laforin polypeptide for use in thepresent disclosure may lack all N-glycosylation sites, relative tonative laforin, or the laforin polypeptide for use in the presentdisclosure may be under-glycosylated, relative to native laforin. Insome embodiments, the laforin polypeptide comprises a modified aminoacid sequence that is unable to be N-glycosylated at one or moreN-glycosylation sites. In some embodiments, asparagine (Asn) of at leastone predicted N-glycosylation site (i.e., a consensus sequencerepresented by the amino acid sequence Asn-Xaa-Ser or Asn-Xaa-Thr) inthe laforin polypeptide is substituted by another amino acid. Thedisclosure contemplates that any one or more of the foregoing examplescan be combined so that a laforin polypeptide of the present disclosurelacks one or more N-glycosylation sites, and thus is either notglycosylated or is under glycosylated relative to native laforin.

In some embodiments, a laforin polypeptide is not O-glycosylated orlacks one or more of the O-glycosylation groups present in a wildtypelaforin polypeptide. In some embodiments, the laforin polypeptidecomprises a modified amino acid sequence that is unable to beO-glycosylated at one or more O-glycosylation sites. In someembodiments, serine or threonine at any one or more predictedO-glycosylation site in the laforin polypeptide sequence is substitutedor deleted. The disclosure contemplates that any one or more of theforegoing examples can be combined so that a laforin polypeptide of thepresent disclosure lacks one or more N-glycosylation and/orO-glycosylation sites, and thus is either not glycosylated or is underglycosylated relative to native laforin.

In one specific embodiment of the present disclosure, a laforinpolypeptide may be modified with nonproteinaceous polymers. In onespecific embodiment, the polymer is polyethylene glycol (“PEG”),polypropylene glycol, or polyoxyalkylenes, in the manner as set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337. PEG is a well-known, water soluble polymer that iscommercially available or can be prepared by ring-opening polymerizationof ethylene glycol according to methods well known in the art (Sandlerand Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages138-161).

By the terms “biological activity”, “bioactivity” or “functional” ismeant the ability of the laforin polypeptide to carry out the functionsassociated with wildtype laforin polypeptides, for example, havingprotein phosphatase activity, glucan phosphatase activity, ability toform a complex with malin, and/or carbohydrate binding activity (e.g.,glycogen binding activity). The terms “biological activity”,“bioactivity”, and “functional” are used interchangeably herein. As usedherein, “fragments” are understood to include bioactive fragments (alsoreferred to as functional fragments) or bioactive variants that exhibit“bioactivity” as described herein. That is, bioactive fragments orvariants of laforin exhibit bioactivity that can be measured and tested.For example, bioactive fragments/functional fragments or variantsexhibit the same or substantially the same bioactivity as native (i.e.,wild-type, or normal) laforin polypeptide, and such bioactivity can beassessed by the ability of the fragment or variant to, e.g., proteinphosphatase activity, glucan phosphatase activity, ability to form acomplex with malin, and/or carbohydrate (e.g., glycogen) bindingactivity. As used herein, “substantially the same” refers to anyparameter (e.g., activity) that is at least 70% of a control againstwhich the parameter is measured. In certain embodiments, “substantiallythe same” also refers to any parameter (e.g., activity) that is at least75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, 100%, 102%, 105%, or 110%of a control against which the parameter is measured. In certainembodiments, fragments or variants of the laforin polypeptide willpreferably retain at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% ofthe laforin biological activity associated with the native laforinpolypeptide, when assessed under the same or substantially the sameconditions.

In certain embodiments, fragments or variants of the laforin polypeptidehave a half-life (t_(1/2)) which is enhanced relative to the half-lifeof the native protein. Preferably, the half-life of laforin fragments orvariants is enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by1000% relative to the half-life of the native laforin polypeptide. Insome embodiments, the protein half-life is determined in vitro, such asin a buffered saline solution or in serum. In other embodiments, theprotein half-life is an in vivo half life, such as the half-life of theprotein in the serum or other bodily fluid of an animal. In addition,fragments or variants can be chemically synthesized using techniquesknown in the art such as conventional Merrifield solid phase f-Moc ort-Boc chemistry. The fragments or variants can be produced(recombinantly or by chemical synthesis) and tested to identify thosefragments or variants that can function as well as or substantiallysimilarly to a native laforin polypeptide.

With respect to methods of increasing laforin bioactivity in cells, thedisclosure contemplates all combinations of any of the foregoing aspectsand embodiments, as well as combinations with any of the embodiments setforth in the detailed description and examples. The described methodsbased on administering chimeric polypeptides or contacting cells withchimeric polypeptides can be performed in vitro (e.g., in cells orculture) or in vivo (e.g., in a patient or animal model). In certainembodiments, the method is an in vitro method. In certain embodiments,the method is an in vivo method.

In some aspects, the present disclosure also provides a method ofproducing any of the foregoing chimeric polypeptides as describedherein. Further, the present disclosure contemplates any number ofcombinations of the foregoing methods and compositions.

In certain aspects, a laforin polypeptide may be a fusion protein whichfurther comprises one or more fusion domains. Well known examples ofsuch fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, and an immunoglobulin heavy chain constant region (Fc),maltose binding protein (MBP), which are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Fusion domains also include “epitopetags,” which are usually short peptide sequences for which a specificantibody is available. Well known epitope tags for which specificmonoclonal antibodies are readily available include FLAG, influenzavirus haemagglutinin (HA), His and c-myc tags. An exemplary His tag hasthe sequence HHHHHH (SEQ ID NO: 7), and an exemplary c-myc tag has thesequence EQKLISEEDL (SEQ ID NO: 8). In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain embodiments, the laforinpolypeptides may contain one or more modifications that are capable ofstabilizing the polypeptides. For example, such modifications enhancethe in vitro half life of the polypeptides, enhance circulatory halflife of the polypeptides or reduce proteolytic degradation of thepolypeptides.

In certain embodiments of any of the foregoing, the laforin portion ofthe chimeric polypeptide of the disclosure comprises a laforinpolypeptide, which in certain embodiments may be a functional fragmentof a laforin polypeptide or may be a substantially full length laforinpolypeptide. In some embodiments, the laforin polypeptide lacks themethionine at the N-terminal-most amino acid position (e.g., lacks themethionine at the first amino acid of any one of SEQ ID NOs: 38 or 39).Suitable laforin polypeptides for use in the chimeric polypeptides andmethods of the disclosure have protein phosphatase activity, glucanphosphatase activity, ability to form a complex with malin, and/orcarbohydrate binding activity (e.g., glycogen binding activity), asevaluated in vitro or in vivo. Exemplary functional fragments comprise,at least 100, 125, 150, 175, 200, 225, 250, 275, 300 or 317 consecutiveamino acid residues of a full length laforin polypeptide (e.g., SEQ IDNOs: 38 or 39). In some embodiments, the functional fragment comprises100-150, 100-200, 100-250, 100-300, 100-330, 200-250, 200-300, 200-330or 300-330 consecutive amino acids of a full-length laforin polypeptide(e.g., SEQ ID NO: 38). In some embodiments, the functional fragmentcomprises 100-150, 100-200, 100-250, 100-300, 100-316, 200-250, 200-300,200-316, or 300-316 consecutive amino acids of a full-length laforinpolypeptide (e.g., SEQ ID NO: 39). Similarly, in certain embodiments,the disclosure contemplates chimeric proteins where the laforin portionis a variant of any of the foregoing laforin polypeptides or bioactivefragments. Exemplary variants have an amino acid sequence at least 90%,92%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acidsequence of a native laforin polypeptide or functional fragment thereof,and such variants retain the laforin variant's protein phosphataseactivity, glucan phosphatase activity, ability to form a complex withmalin, and/or glycogen binding activity. The disclosure contemplateschimeric polypeptides and the use of such polypeptides wherein thelaforin portion comprises any of the laforin polypeptides, fragments, orvariants described herein in combination with any internalizing moietydescribed herein. Moreover, in certain embodiments, the laforin portionof any of the foregoing chimeric polypeptides may, in certainembodiments, by a fusion protein. Any such chimeric polypeptidescomprising any combination of laforin portions and internalizing moietyportions, and optionally including one or more linkers, one or moretags, etc., may be used in any of the methods of the disclosure.

III. AGL Polypeptides

In certain embodiments, the non-internalizing moiety polypeptide portionof a chimeric polypeptide of the disclosure (or a chimeric polypeptidefor use in the methods of the disclosure) is an AGL polypeptide. Inother words, in certain embodiments, AGL-containing chimericpolypeptides are provided. Exemplary AGL polypeptides for use in themethods and compositions of the disclosure are provided herein.

As used herein, the AGL polypeptides include various functionalfragments and variants, fusion proteins, and modified forms of thewildtype AGL polypeptide. Such functional fragments or variants, fusionproteins, and modified forms of the AGL polypeptides have at least aportion of the amino acid sequence of substantial sequence identity tothe native AGL protein, and retain the function of the native AGLprotein (e.g., retain the two enzymatic activities of native AGL). Itshould be noted that “retain the function” does not mean that theactivity of a particular fragment must be identical or substantiallyidentical to that of the native protein although, in some embodiments,it may be. However, to retain the native activity, that native activityshould be at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% that of the nativeprotein to which such activity is being compared, with the comparisonbeing made under the same or similar conditions. In some embodiments,retaining the native activity may include scenarios in which a fragmentor variant has improved activity versus the native protein to which suchactivity is being compared, e.g., at least 105%, at least 110%, at least120%, or at least 125%, with the comparison being bade under the same orsimilar conditions.

In certain embodiments, a functional fragment, variant, or fusionprotein of an AGL polypeptide comprises an amino acid sequence that isat least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an AGLpolypeptide (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to SEQ ID NOs: 40-42), or fragments thereof.

In certain embodiments, the AGL polypeptide for use in the chimericpolypeptides and methods of the disclosure is a full length orsubstantially full length AGL polypeptide. In certain embodiments, theAGL polypeptide for use in the chimeric polypeptide and methods of thedisclosure is a functional fragment that has amylo-1,6-glucosidaseactivity and 4-alpha-glucotransferase activity.

In certain embodiments, fragments or variants of the AGL polypeptidescan be obtained by screening polypeptides recombinantly produced fromthe corresponding fragment of the nucleic acid encoding an AGLpolypeptide. In addition, fragments or variants can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. The fragments orvariants can be produced (recombinantly or by chemical synthesis) andtested to identify those fragments or variants that can function as anative AGL protein, for example, by testing their ability to treatForbes-Cori Disease in vivo and/or by confirming in vitro (e.g., in acell free or cell based assay) that the fragment or variant hasamylo-1,6-glucosidase activity and 4-alpha-glucotransferase activity. Anexample of an in vitro assay for testing for activity of the AGLpolypeptides disclosed herein would be to treat Forbes-Cori cells withor without the AGL-containing chimeric polypeptides and then, after aperiod of incubation, stain the cells for the presence of glycogen,e.g., by using a periodic acid Schiff (PAS) stain.

In certain embodiments, the present disclosure contemplates modifyingthe structure of an AGL polypeptide for such purposes as enhancingtherapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelflife and resistance to proteolytic degradation in vivo). Modifiedpolypeptides can be produced, for instance, by amino acid substitution,deletion, or addition. For instance, it is reasonable to expect, forexample, that an isolated replacement of a leucine with an isoleucine orvaline, an aspartate with a glutamate, a threonine with a serine, or asimilar replacement of an amino acid with a structurally related aminoacid (e.g., conservative mutations) will not have a major effect on theAGL biological activity of the resulting molecule. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains.

This disclosure further contemplates generating sets of combinatorialmutants of an AGL polypeptide, as well as truncation mutants, and isespecially useful for identifying functional variant sequences.Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring AGL polypeptide. Likewise,mutagenesis can give rise to variants which have intracellularhalf-lives dramatically different than the corresponding wild-type AGLpolypeptide. For example, the altered protein can be rendered eithermore stable or less stable to proteolytic degradation or other cellularprocess which result in destruction of, or otherwise inactivation ofAGL. Such variants can be utilized to alter the AGL polypeptide level bymodulating their half-life. There are many ways by which the library ofpotential AGL variants sequences can be generated, for example, from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be carried out in an automatic DNA synthesizer, andthe synthetic genes then be ligated into an appropriate gene forexpression. The purpose of a degenerate set of genes is to provide, inone mixture, all of the sequences encoding the desired set of potentialpolypeptide sequences. The synthesis of degenerate oligonucleotides iswell known in the art (see for example, Narang, S A (1983) Tetrahedron39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd ClevelandSympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier pp 273-289;Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al.,(1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477).Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al., (1990) Science 249:386-390;Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990)Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, AGL polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of the AGL polypeptide.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of the AGL polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

In certain embodiments, an AGL polypeptide may include a peptidomimetic.As used herein, the term “peptidomimetic” includes chemically modifiedpeptides and peptide-like molecules that contain non-naturally occurringamino acids, peptoids, and the like. Peptidomimetics provide variousadvantages over a peptide, including enhanced stability whenadministered to a subject. Methods for identifying a peptidomimetic arewell known in the art and include the screening of databases thatcontain libraries of potential peptidomimetics. For example, theCambridge Structural Database contains a collection of greater than300,000 compounds that have known crystal structures (Allen et al., ActaCrystallogr. Section B, 35:2331 (1979)). Where no crystal structure of atarget molecule is available, a structure can be generated using, forexample, the program CONCORD (Rusinko et al., J. Chem. Inf. Comput. Sci.29:251 (1989)). Another database, the Available Chemicals Directory(Molecular Design Limited, Informations Systems; San Leandro Calif.),contains about 100,000 compounds that are commercially available andalso can be searched to identify potential peptidomimetics of the AGLpolypeptides.

In certain embodiments, an AGL polypeptide may further comprisepost-translational modifications. Exemplary post-translational proteinmodifications include phosphorylation, acetylation, methylation,ADP-ribosylation, ubiquitination, glycosylation, carbonylation,sumoylation, biotinylation or addition of a polypeptide side chain or ofa hydrophobic group. As a result, the modified AGL polypeptides maycontain non-amino acid elements, such as lipids, poly- ormono-saccharides, and phosphates. Effects of such non-amino acidelements on the functionality of an AGL polypeptide may be tested forits biological activity, for example, its ability to hydrolyze glycogenor treat Forbes-Cori Disease. In certain embodiments, the AGLpolypeptide may further comprise one or more polypeptide portions thatenhance one or more of in vivo stability, in vivo half life,uptake/administration, and/or purification. In other embodiments, theinternalizing moiety comprises an antibody or an antigen-bindingfragment thereof.

In some embodiments, an AGL polypeptide is not N-glycosylated or lacksone or more of the N-glycosylation groups present in a wildtype AGLpolypeptide. For example, the AGL polypeptide for use in the presentdisclosure may lack all N-glycosylation sites, relative to native AGL,or the AGL polypeptide for use in the present disclosure may beunder-glycosylated, relative to native AGL. In some embodiments, the AGLpolypeptide comprises a modified amino acid sequence that is unable tobe N-glycosylated at one or more N-glycosylation sites. In someembodiments, asparagine (Asn) of at least one predicted N-glycosylationsite (i.e., a consensus sequence represented by the amino acid sequenceAsn-Xaa-Ser or Asn-Xaa-Thr) in the AGL polypeptide is substituted byanother amino acid. Examples of Asn-Xaa-Ser sequence stretches in theAGL amino acid sequence include amino acids corresponding to amino acidpositions 813-815, 839-841, 927-929, and 1032-1034 of SEQ ID NO: 40.Examples of Asn-Xaa-Thr sequence stretches in the AGL amino acidsequence include amino acids corresponding to amino acid positions69-71, 219-221, 797-799, 1236-1238 and 1380-1382. In some embodiments,the asparagine at any one, or combination, of amino acid positionscorresponding to amino acid positions 69, 219, 797, 813, 839, 927, 1032,1236 and 1380 of SEQ ID NO: 40 is substituted or deleted. In someembodiments, the serine at any one, or combination of, amino acidpositions corresponding to amino acid positions 815, 841, 929 and 1034of SEQ ID NO: 40 is substituted or deleted. In some embodiments, thethreonine at any one, or combination of, amino acid positionscorresponding to amino acid positions 71, 221, 799, 1238 and 1382 of SEQID NO: 40 is substituted or deleted. In some embodiments, the Xaa aminoacid corresponding to any one of, or combination of, amino acidpositions 220, 798, 814, 840, 928, 1033, 1237 and 1381 of SEQ ID NO: 40is deleted or replaced with a proline. The disclosure contemplates thatany one or more of the foregoing examples can be combined so that an AGLpolypeptide of the present disclosure lacks one or more N-glycosylationsites, and thus is either not glycosylated or is under glycosylatedrelative to native AGL.

In some embodiments, an AGL polypeptide is not O-glycosylated or lacksone or more of the O-glycosylation groups present in a wildtype AGLpolypeptide. In some embodiments, the AGL polypeptide comprises amodified amino acid sequence that is unable to be O-glycosylated at oneor more O-glycosylation sites. In some embodiments, serine or threonineat any one or more predicted O-glycosylation site in the AGL polypeptidesequence is substituted or deleted. The disclosure contemplates that anyone or more of the foregoing examples can be combined so that an AGLpolypeptide of the present disclosure lacks one or more N-glycosylationand/or O-glycosylation sites, and thus is either not glycosylated or isunder glycosylated relative to native AGL.

In one specific embodiment of the present disclosure, an AGL polypeptidemay be modified with nonproteinaceous polymers. In one specificembodiment, the polymer is polyethylene glycol (“PEG”), polypropyleneglycol, or polyoxyalkylenes, in the manner as set forth in U.S. Pat.Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161).

By the terms “biological activity”, “bioactivity” or “functional” ismeant the ability of the AGL protein to carry out the functionsassociated with wildtype AGL proteins, for example, havingoligo-1,4-1,4-glucotransferase activity and/or amylo-1,6-glucosidaseactivity. The terms “biological activity”, “bioactivity”, and“functional” are used interchangeably herein. As used herein,“fragments” are understood to include bioactive fragments (also referredto as functional fragments) or bioactive variants that exhibit“bioactivity” as described herein. That is, bioactive fragments orvariants of AGL exhibit bioactivity that can be measured and tested. Forexample, bioactive fragments/functional fragments or variants exhibitthe same or substantially the same bioactivity as native (i.e.,wild-type, or normal) AGL protein, and such bioactivity can be assessedby the ability of the fragment or variant to, e.g., debranch glycogenvia the AGL fragment's or variant's 4-alpha-glucotransferase activityand/or amylo-1,6-glucosidase activity. As used herein, “substantiallythe same” refers to any parameter (e.g., activity) that is at least 70%of a control against which the parameter is measured. In certainembodiments, “substantially the same” also refers to any parameter(e.g., activity) that is at least 75%, 80%, 85%, 90%, 92%, 95%, 97%,98%, 99%, 100%, 102%, 105%, or 110% of a control against which theparameter is measured. In certain embodiments, fragments or variants ofthe AGL polypeptide will preferably retain at least 50%, 60%, 70%, 80%,85%, 90%, 95% or 100% of the AGL biological activity associated with thenative AGL polypeptide, when assessed under the same or substantiallythe same conditions.

In certain embodiments, fragments or variants of the AGL polypeptidehave a half-life (t_(1/2)) which is enhanced relative to the half-lifeof the native protein. Preferably, the half-life of AGL fragments orvariants is enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by1000% relative to the half-life of the native AGL protein. In someembodiments, the protein half-life is determined in vitro, such as in abuffered saline solution or in serum. In other embodiments, the proteinhalf-life is an in vivo half life, such as the half-life of the proteinin the serum or other bodily fluid of an animal. In addition, fragmentsor variants can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. The fragments or variants can be produced (recombinantly orby chemical synthesis) and tested to identify those fragments orvariants that can function as well as or substantially similarly to anative AGL protein.

With respect to methods of increasing AGL bioactivity in cells, thedisclosure contemplates all combinations of any of the foregoing aspectsand embodiments, as well as combinations with any of the embodiments setforth in the detailed description and examples. The described methodsbased on administering chimeric polypeptides or contacting cells withchimeric polypeptides can be performed in vitro (e.g., in cells orculture) or in vivo (e.g., in a patient or animal model). In certainembodiments, the method is an in vitro method. In certain embodiments,the method is an in vivo method.

In some aspects, the present disclosure also provides a method ofproducing any of the foregoing chimeric polypeptides as describedherein. Further, the present disclosure contemplates any number ofcombinations of the foregoing methods and compositions.

In certain aspects, an AGL polypeptide may be a fusion protein whichfurther comprises one or more fusion domains. Well known examples ofsuch fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, and an immunoglobulin heavy chain constant region (Fc),maltose binding protein (MBP), which are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Fusion domains also include “epitopetags,” which are usually short peptide sequences for which a specificantibody is available. Well known epitope tags for which specificmonoclonal antibodies are readily available include FLAG, influenzavirus haemagglutinin (HA), His and c-myc tags. An exemplary His tag hasthe sequence HHHHHH (SEQ ID NO: 7), and an exemplary c-myc tag has thesequence EQKLISEEDL (SEQ ID NO: 8). In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain embodiments, the AGL polypeptidesmay contain one or more modifications that are capable of stabilizingthe polypeptides. For example, such modifications enhance the in vitrohalf life of the polypeptides, enhance circulatory half life of thepolypeptides or reduce proteolytic degradation of the polypeptides.

In certain embodiments of any of the foregoing, the AGL portion of thechimeric polypeptide of the disclosure comprises an AGL polypeptide,which in certain embodiments may be a functional fragment of an AGLpolypeptide or may be a substantially full length AGL polypeptide. Insome embodiments, the AGL polypeptide lacks the methionine at theN-terminal-most amino acid position (e.g., lacks the methionine at thefirst amino acid of any one of SEQ ID NOs: 40-42). Suitable AGLpolypeptides for use in the chimeric polypeptides and methods of thedisclosure have oligo-1,4-1,4-glucotransferase activity andamylo-1,6-glucosidase activity, as evaluated in vitro or in vivo.Exemplary functional fragments comprise, at least 500, at least 525, atleast 550, at least 575, at least 600, at least 625, at least 650, atleast 675, at least 700, at least 725, at least 750, at least 775, atleast 800, at least 825, at least 850, at least 875, at least 900, atleast 925, at least 925, at least 950, at least 975, at least 1000, atleast 1025, at least 1050, at least 1075, at least 1100, at least 1125,at least 1150, at least 1175, at least 1200, at least 1225, at least1250, at least 1275, at least 1300, at least 1325, at least 1350, atleast 1375, at least 1400, at least 1425, at least 1450, at least 1475,at least 1500, at least 1525 or at least 1532 amino consecutive aminoacid residues of a full length AGL polypeptide (e.g., SEQ ID NOs:40-42). In some embodiments, the functional fragment comprises 500-750,500-1000, 500-1200, 500-1300, 500-1500, 1000-1100, 1000-1200, 1000-1300,1000-1400, 1000-1500, 1000-1532 consecutive amino acids of a full-lengthAGL polypeptide (e.g., SEQ ID NOs: 40-42). Similarly, in certainembodiments, the disclosure contemplates chimeric proteins where the AGLportion is a variant of any of the foregoing AGL polypeptides orbioactive fragments. Exemplary variants have an amino acid sequence atleast 90%, 92%, 95%, 96%, 97%, 98%, or at least 99% identical to theamino acid sequence of a native AGL polypeptide or functional fragmentthereof, and such variants retain the ability to debranch glycogen viathe AGL variant's oligo-1,4-1,4-glucotransferase activity andamylo-1,6-glucosidase activity. The disclosure contemplates chimericpolypeptides and the use of such polypeptides wherein the AGL portioncomprises any of the AGL polypeptides, fragments, or variants describedherein in combination with any internalizing moiety described herein.Moreover, in certain embodiments, the AGL portion of any of theforegoing chimeric polypeptides may, in certain embodiments, by a fusionprotein. Any such chimeric polypeptides comprising any combination ofAGL portions and internalizing moiety portions, and optionally includingone or more linkers, one or more tags, etc., may be used in any of themethods of the disclosure.

IV. Malin Polypeptides

In certain embodiments, the non-internalizing moiety polypeptide portionof a chimeric polypeptide of the disclosure (or a chimeric polypeptidefor use in the methods of the disclosure) is malin polypeptide. In otherwords, in certain embodiments, malin-containing chimeric polypeptidesare provided. Exemplary malin polypeptides for use in the methods andcompositions of the disclosure are provided herein.

As used herein, the malin polypeptides include various functionalfragments and variants, fusion proteins, and modified forms of thewildtype malin polypeptide. Such functional fragments or variants,fusion proteins, and modified forms of the malin polypeptides have atleast a portion of the amino acid sequence of substantial sequenceidentity to the native malin polypeptide, and retain the function of thenative malin polypeptide (e.g., E3 ubiquitin ligase activity and/orability to form a complex with laforin). It should be noted that “retainthe function” does not mean that the activity of a particular fragmentmust be identical or substantially identical to that of the nativeprotein although, in some embodiments, it may be. However, to retain thenative activity, that native activity should be at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% that of the native protein to which such activity isbeing compared, with the comparison being made under the same or similarconditions. In some embodiments, retaining the native activity mayinclude scenarios in which a fragment or variant has improved activityversus the native protein to which such activity is being compared,e.g., at least 105%, at least 110%, at least 120%, or at least 125%,with the comparison being bade under the same or similar conditions.

Wildtype malin polypeptide has two functional domains: a RING finger E3ubiquitin ligase domain and six repeats of NHL that are defined by (andnamed after) amino acid sequence homologies with NCL-1, HT2A and LIN41proteins. In some embodiments, the malin polypeptide or functionalfragment or variant thereof comprises a functional RING finger E3ubiquitin ligase domain and/or at least 1, 2, 3, 4, 5 or all six NHLrepeats. In some embodiments, the malin polypeptide or functionalfragment or variant thereof comprises the functional RING finger E3ubiquitin ligase domain and all six NHL repeats.

In certain embodiments, a malin polypeptide, functional fragment,variant, or fusion protein of a malin polypeptide comprises an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to a malin polypeptide (e.g., at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to SEQ ID NO: 43), or fragments thereof.

In certain embodiments, the malin polypeptide for use in the chimericpolypeptides and methods of the disclosure is a full length orsubstantially full length malin polypeptide. In certain embodiments, themalin polypeptide for use in the chimeric polypeptide and methods of thedisclosure is a functional fragment that has E3 ubiquitin ligaseactivity and/or the ability to form a complex with laforin. In certainembodiments of any of the foregoing, the malin polypeptide optionallyincludes (or excludes) the N-terminal methionine.

In certain embodiments, fragments or variants of the malin polypeptidescan be obtained by screening polypeptides recombinantly produced fromthe corresponding fragment of the nucleic acid encoding a malinpolypeptide. In addition, fragments or variants can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. The fragments orvariants can be produced (recombinantly or by chemical synthesis) andtested to identify those fragments or variants that can function as anative malin polypeptide, for example, by testing their ability to treatLafora Disease in vivo and/or by confirming in vitro (e.g., in a cellfree or cell based assay) that the fragment or variant has protein E3ubiquitin ligase activity and/or ability to form a complex with laforin.An example of an in vitro assay for testing for activity of the malinpolypeptides disclosed herein would be to test for malin's ability toubiquitinate a protein substrate in vitro.

In certain embodiments, the present disclosure contemplates modifyingthe structure of a malin polypeptide for such purposes as enhancingtherapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelflife and resistance to proteolytic degradation in vivo). Modifiedpolypeptides can be produced, for instance, by amino acid substitution,deletion, or addition. For instance, it is reasonable to expect, forexample, that an isolated replacement of a leucine with an isoleucine orvaline, an aspartate with a glutamate, a threonine with a serine, or asimilar replacement of an amino acid with a structurally related aminoacid (e.g., conservative mutations) will not have a major effect on themalin biological activity of the resulting molecule. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains.

This disclosure further contemplates generating sets of combinatorialmutants of a malin polypeptide, as well as truncation mutants, and isespecially useful for identifying functional variant sequences.Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring malin polypeptide. Likewise,mutagenesis can give rise to variants which have intracellularhalf-lives dramatically different than the corresponding wild-type malinpolypeptide. For example, the altered protein can be rendered eithermore stable or less stable to proteolytic degradation or other cellularprocess which result in destruction of, or otherwise inactivation ofmalin. Such variants can be utilized to alter the malin polypeptidelevel by modulating their half-life. There are many ways by which thelibrary of potential malin variants sequences can be generated, forexample, from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes then can be ligated into anappropriate gene for expression. The purpose of a degenerate set ofgenes is to provide, in one mixture, all of the sequences encoding thedesired set of potential polypeptide sequences. The synthesis ofdegenerate oligonucleotides is well known in the art (see for example,Narang, S A (1983) Tetrahedron 39:3; Itakura et al., (1981) RecombinantDNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton,Amsterdam: Elsevier pp273-289; Itakura et al., (1984) Annu. Rev.Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al.,(1983)

Nucleic Acid Res. 11:477). Such techniques have been employed in thedirected evolution of other proteins (see, for example, Scott et al.,(1990) Science 249:386-390; Roberts et al., (1992) PNAS USA89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al.,(1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, malin polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J.

Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem.268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838; andCunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of the malin polypeptide.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of the malin polypeptides. The most widelyused techniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

In certain embodiments, a malin polypeptide may include apeptidomimetic. As used herein, the term “peptidomimetic” includeschemically modified peptides and peptide-like molecules that containnon-naturally occurring amino acids, peptoids, and the like.Peptidomimetics provide various advantages over a peptide, includingenhanced stability when administered to a subject. Methods foridentifying a peptidomimetic are well known in the art and include thescreening of databases that contain libraries of potentialpeptidomimetics. For example, the Cambridge Structural Database containsa collection of greater than 300,000 compounds that have known crystalstructures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)).Where no crystal structure of a target molecule is available, astructure can be generated using, for example, the program CONCORD(Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Anotherdatabase, the Available Chemicals Directory (Molecular Design Limited,Informations Systems; San Leandro Calif.), contains about 100,000compounds that are commercially available and also can be searched toidentify potential peptidomimetics of the malin polypeptides.

In certain embodiments, a malin polypeptide may further comprisepost-translational modifications. Exemplary post-translational proteinmodifications include phosphorylation, acetylation, methylation,ADP-ribosylation, ubiquitination, glycosylation, carbonylation,sumoylation, biotinylation or addition of a polypeptide side chain or ofa hydrophobic group. As a result, the modified malin polypeptides maycontain non-amino acid elements, such as lipids, poly- ormono-saccharides, and phosphates. Effects of such non-amino acidelements on the functionality of a malin polypeptide may be tested forits biological activity, for example, its retention of E3 ubiquitinligase activity and/or ability to form a complex with laforin and/or itsability to treat Lafora Disease. In certain embodiments, the malinpolypeptide may further comprise one or more polypeptide portions thatenhance one or more of in vivo stability, in vivo half life,uptake/administration, and/or purification. In other embodiments, theinternalizing moiety comprises an antibody or an antigen-bindingfragment thereof.

In some embodiments, a malin polypeptide is not N-glycosylated or lacksone or more of the N-glycosylation groups present in a wildtype malinpolypeptide. For example, the malin polypeptide for use in the presentdisclosure may lack all N-glycosylation sites, relative to native malin,or the malin polypeptide for use in the present disclosure may beunder-glycosylated, relative to native malin. In some embodiments, themalin polypeptide comprises a modified amino acid sequence that isunable to be N-glycosylated at one or more N-glycosylation sites. Insome embodiments, asparagine (Asn) of at least one predictedN-glycosylation site (i.e., a consensus sequence represented by theamino acid sequence Asn-Xaa-Ser or Asn-Xaa-Thr) in the malin polypeptideis substituted by another amino acid. The disclosure contemplates thatany one or more of the foregoing examples can be combined so that amalin polypeptide of the present disclosure lacks one or moreN-glycosylation sites, and thus is either not glycosylated or is underglycosylated relative to native malin.

In some embodiments, a malin polypeptide is not O-glycosylated or lacksone or more of the O-glycosylation groups present in a wildtype malinpolypeptide. In some embodiments, the malin polypeptide comprises amodified amino acid sequence that is unable to be O-glycosylated at oneor more O-glycosylation sites. In some embodiments, serine or threonineat any one or more predicted O-glycosylation site in the malinpolypeptide sequence is substituted or deleted. The disclosurecontemplates that any one or more of the foregoing examples can becombined so that a malin polypeptide of the present disclosure lacks oneor more N-glycosylation and/or O-glycosylation sites, and thus is eithernot glycosylated or is under glycosylated relative to native malin.

In one specific embodiment of the present disclosure, a malinpolypeptide may be modified with nonproteinaceous polymers. In onespecific embodiment, the polymer is polyethylene glycol (“PEG”),polypropylene glycol, or polyoxyalkylenes, in the manner as set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337. PEG is a well-known, water soluble polymer that iscommercially available or can be prepared by ring-opening polymerizationof ethylene glycol according to methods well known in the art (Sandlerand Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages138-161).

By the terms “biological activity”, “bioactivity” or “functional” ismeant the ability of the malin polypeptide to carry out the functionsassociated with wildtype malin polypeptides, for example, E3 ubiquitinligase activity and/or ability to form a complex with laforin. The terms“biological activity”, “bioactivity”, and “functional” are usedinterchangeably herein. As used herein, “fragments” are understood toinclude bioactive fragments (also referred to as functional fragments)or bioactive variants that exhibit “bioactivity” as described herein.That is, bioactive fragments or variants of malin exhibit bioactivitythat can be measured and tested. For example, bioactivefragments/functional fragments or variants exhibit the same orsubstantially the same bioactivity as native (i.e., wild-type, ornormal) malin polypeptide, and such bioactivity can be assessed by theability of the fragment or variant to, e.g., E3 ubiquitin ligaseactivity and/or ability to form a complex with laforin. As used herein,“substantially the same” refers to any parameter (e.g., activity) thatis at least 70% of a control against which the parameter is measured. Incertain embodiments, “substantially the same” also refers to anyparameter (e.g., activity) that is at least 75%, 80%, 85%, 90%, 92%,95%, 97%, 98%, 99%, 100%, 102%, 105%, or 110% of a control against whichthe parameter is measured. In certain embodiments, fragments or variantsof the malin polypeptide will preferably retain at least 50%, 60%, 70%,80%, 85%, 90%, 95% or 100% of the malin biological activity associatedwith the native malin polypeptide, when assessed under the same orsubstantially the same conditions.

In certain embodiments, fragments or variants of the malin polypeptidehave a half-life (t_(1/2)) which is enhanced relative to the half-lifeof the native protein. Preferably, the half-life of malin fragments orvariants is enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by1000% relative to the half-life of the native malin polypeptide. In someembodiments, the protein half-life is determined in vitro, such as in abuffered saline solution or in serum. In other embodiments, the proteinhalf-life is an in vivo half life, such as the half-life of the proteinin the serum or other bodily fluid of an animal. In addition, fragmentsor variants can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. The fragments or variants can be produced (recombinantly orby chemical synthesis) and tested to identify those fragments orvariants that can function as well as or substantially similarly to anative malin polypeptide.

With respect to methods of increasing malin bioactivity in cells, thedisclosure contemplates all combinations of any of the foregoing aspectsand embodiments, as well as combinations with any of the embodiments setforth in the detailed description and examples. The described methodsbased on administering chimeric polypeptides or contacting cells withchimeric polypeptides can be performed in vitro (e.g., in cells orculture) or in vivo (e.g., in a patient or animal model). In certainembodiments, the method is an in vitro method. In certain embodiments,the method is an in vivo method.

In some aspects, the present disclosure also provides a method ofproducing any of the foregoing chimeric polypeptides as describedherein. Further, the present disclosure contemplates any number ofcombinations of the foregoing methods and compositions.

In certain aspects, a malin polypeptide may be a fusion protein whichfurther comprises one or more fusion domains. Well known examples ofsuch fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, and an immunoglobulin heavy chain constant region (Fc),maltose binding protein (MBP), which are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Fusion domains also include “epitopetags,” which are usually short peptide sequences for which a specificantibody is available. Well known epitope tags for which specificmonoclonal antibodies are readily available include FLAG, influenzavirus haemagglutinin (HA), His and c-myc tags. An exemplary His tag hasthe sequence HHHHHH (SEQ ID NO: 7), and an exemplary c-myc tag has thesequence EQKLISEEDL (SEQ ID NO: 8). In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain embodiments, the malinpolypeptides may contain one or more modifications that are capable ofstabilizing the polypeptides. For example, such modifications enhancethe in vitro half life of the polypeptides, enhance circulatory halflife of the polypeptides or reduce proteolytic degradation of thepolypeptides.

In certain embodiments of any of the foregoing, the malin portion of thechimeric polypeptide of the disclosure comprises a malin polypeptide,which in certain embodiments may be a functional fragment of a malinpolypeptide or may be a substantially full length malin polypeptide. Insome embodiments, the malin polypeptide lacks the methionine at theN-terminal-most amino acid position (e.g., lacks the methionine at thefirst amino acid of SEQ ID NO: 43). Suitable malin polypeptides for usein the chimeric polypeptides and methods of the disclosure have E3ubiquitin ligase activity and/or the ability to form a complex withlaforin, as evaluated in vitro or in vivo. Exemplary functionalfragments comprise, at least 100, 125, 150, 175, 200, 225, 250, 275,300, 350, 370, 380, 390, or 395 consecutive amino acid residues of afull length malin polypeptide (e.g., SEQ ID NO: 43). In someembodiments, the functional fragment comprises 100-150, 100-200,100-250, 100-300, 100-395, 200-250, 200-300, 200-395, 300-395, 350-395or 380-395, 390-395 consecutive amino acids of a full-length malinpolypeptide (e.g., SEQ ID NO: 43). Similarly, in certain embodiments,the disclosure contemplates chimeric proteins where the malin portion isa variant of any of the foregoing malin polypeptides or bioactivefragments. Exemplary variants have an amino acid sequence at least 90%,92%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acidsequence of a native malin polypeptide or functional fragment thereof,and such variants have E3 ubiquitin ligase activity and/or the abilityto form a complex with laforin. The disclosure contemplates chimericpolypeptides and the use of such polypeptides wherein the malin portioncomprises any of the malin polypeptides, fragments, or variantsdescribed herein in combination with any internalizing moiety describedherein. Moreover, in certain embodiments, the malin portion of any ofthe foregoing chimeric polypeptides may, in certain embodiments, by afusion protein. Any such chimeric polypeptides comprising anycombination of malin portions and internalizing moiety portions, andoptionally including one or more linkers, one or more tags, etc., may beused in any of the methods of the disclosure.

V. Alpha-Amylase Polypeptides

In certain embodiments, the non-internalizing moiety polypeptide portionof a chimeric polypeptide of the disclosure (or a chimeric polypeptidefor use in the methods of the disclosure) is an alpha-amylasepolypeptide (e.g., a salivary or pancreatic alpha-amylase). In otherwords, in certain embodiments, alpha-amylase-containing chimericpolypeptides are provided. Exemplary alpha-amylase polypeptides for usein the methods and compositions of the disclosure are provided herein.In some embodiments, the alpha-amylase polypeptides have utility inclearing excess glycogen in diseased cells. In some embodiments, thediseased cells are the cells of a subject having a glycogen storagedisease or a glycogen metabolic disorder. In some embodiments, thediseased cells are from a subject having Pompe Disease, AndersenDisease, von Gierke Disease, Lafora Disease and/or Forbes-Cori Disease.In some embodiments, the diseased cells are from a subject having LaforaDisease and/or Forbes-Cori Disease.

In certain embodiments, any of the alpha-amylase polypeptides referredto herein may be substituted with a gamma-amylase. In certainembodiments, the gamma-amylase is capable of catalyzing the hydrolysisof terminal 1,4-linked alpha-D-glucose residues successively fromnon-reducing ends of polysaccharide chains with the release ofbeta-glucose. In some embodiments, the gamma-amylase is also able tohydrolyze 1,6-alpha-glucosidic bonds when the next bond in sequence is1,4 in a glycogen molecule.

In some embodiments, the alpha-amylase is a monomer. In someembodiments, the alpha-amylase is a dimer or a trimer. In someembodiments, the alpha-amylase has been mutated such that it isincapable of multimerizing (e.g., the alpha-amylase has been mutatedsuch that it is incapable of dimerizing or trimerizing). In someembodiments, the alpha-amylase has been treated with an agent thatinhibits multimerization (e.g., dimerization or trimerization) of thealpha-amylase. In some embodiments, the agent is a small molecule.

As used herein, the alpha-amylase polypeptides include variousfunctional fragments and variants, fusion proteins, and modified formsof the wildtype alpha-amylase polypeptide. In certain embodiments, thealpha-amylase or fragment or variant thereof is a salivary alpha-amylaseor fragment or variant thereof. In certain embodiments, thealpha-amylase or fragment or variant thereof is a pancreaticalpha-amylase or fragment or variant thereof. In certain embodiments,the alpha-amylase or fragment or variant thereof is a mammalian alpha-amylase or fragment or variant thereof. In particular embodiments, thealpha-amylase or fragment or variant thereof is a human alpha- amylaseor fragment or variant thereof. Such functional fragments or variants,fusion proteins, and modified forms of the alpha-amylase polypeptideshave at least a portion of the amino acid sequence of substantialsequence identity to the native alpha-amylase polypeptide, and retainthe function of the native alpha-amylase polypeptide (e.g., ability tohydrolyze alpha-1,4-glucosidic bonds). It should be noted that “retainthe function” does not mean that the activity of a particular fragmentmust be identical or substantially identical to that of the nativeprotein although, in some embodiments, it may be. However, to retain thenative activity, that native activity should be at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% that of the native protein to which such activity isbeing compared, with the comparison being made under the same or similarconditions. In some embodiments, retaining the native activity mayinclude scenarios in which a fragment or variant has improved activityversus the native protein to which such activity is being compared,e.g., at least 105%, at least 110%, at least 120%, or at least 125%,with the comparison being bade under the same or similar conditions.

In certain embodiments, a functional fragment, variant, or fusionprotein of an alpha-amylase polypeptide comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical toan alpha-amylase polypeptide (e.g., at least 80%, 85%, 90%, 95%, 97%,98%, 99% or 100% identical to SEQ ID NO: 44 or 45), or fragmentsthereof.

In certain embodiments, the alpha-amylase polypeptide for use in thechimeric polypeptides and methods of the disclosure is a full length orsubstantially full length alpha-amylase polypeptide. In certainembodiments, the alpha-amylase polypeptide for use in the chimericpolypeptide and methods of the disclosure is a functional fragment thathas alpha-1,4-glucosidic bond hydrolytic activity.

In certain embodiments, fragments or variants of the alpha-amylasepolypeptides can be obtained by screening polypeptides recombinantlyproduced from the corresponding fragment of the nucleic acid encoding analpha-amylase polypeptide. In addition, fragments or variants can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments or variants can be produced (recombinantly or by chemicalsynthesis) and tested to identify those fragments or variants that canfunction as a native alpha-amylase polypeptide, for example, by testingtheir ability to treat Lafora Disease in vivo and/or by confirming invitro (e.g., in a cell free or cell based assay) that the fragment orvariant has alpha-1,4-glucosidic bond hydrolytic activity. An example ofan in vitro assay for testing for activity of the alpha-amylasepolypeptides disclosed herein would be to treat Lafora cells with orwithout the alpha-amylase-containing chimeric polypeptides and then,after a period of incubation, examining levels of polyglucosan.

In certain embodiments, the present disclosure contemplates modifyingthe structure of an alpha-amylase polypeptide for such purposes asenhancing therapeutic or prophylactic efficacy, or stability (e.g., exvivo shelf life and resistance to proteolytic degradation in vivo).Modified polypeptides can be produced, for instance, by amino acidsubstitution, deletion, or addition. For instance, it is reasonable toexpect, for example, that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the alpha-amylase biological activity of the resultingmolecule. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains.

This disclosure further contemplates generating sets of combinatorialmutants of an alpha-amylase polypeptide, as well as truncation mutants,and is especially useful for identifying functional variant sequences.Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring alpha-amylase polypeptide.Likewise, mutagenesis can give rise to variants which have intracellularhalf-lives dramatically different than the corresponding wild-typealpha-amylase polypeptide. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular process which result in destruction of, or otherwiseinactivation of alpha-amylase. Such variants can be utilized to alterthe alpha-amylase polypeptide level by modulating their half-life. Thereare many ways by which the library of potential alpha-amylase variantssequences can be generated, for example, from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be carried out in an automatic DNA synthesizer, and thesynthetic genes then can be ligated into an appropriate gene forexpression. The purpose of a degenerate set of genes is to provide, inone mixture, all of the sequences encoding the desired set of potentialpolypeptide sequences. The synthesis of degenerate oligonucleotides iswell known in the art (see for example, Narang, S A (1983) Tetrahedron39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd ClevelandSympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier pp273-289;Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al.,(1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477).Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al., (1990) Science 249:386-390;Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990)Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, alpha-amylase polypeptide variantscan be generated and isolated from a library by screening using, forexample, alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of the alpha-amylase polypeptide.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of the alpha-amylase polypeptides. The mostwidely used techniques for screening large gene libraries typicallycomprises cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

In certain embodiments, an alpha-amylase polypeptide may include apeptidomimetic. As used herein, the term “peptidomimetic” includeschemically modified peptides and peptide-like molecules that containnon-naturally occurring amino acids, peptoids, and the like.Peptidomimetics provide various advantages over a peptide, includingenhanced stability when administered to a subject. Methods foridentifying a peptidomimetic are well known in the art and include thescreening of databases that contain libraries of potentialpeptidomimetics. For example, the Cambridge Structural Database containsa collection of greater than 300,000 compounds that have known crystalstructures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)).Where no crystal structure of a target molecule is available, astructure can be generated using, for example, the program CONCORD(Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Anotherdatabase, the Available Chemicals Directory (Molecular Design Limited,Informations Systems; San Leandro Calif.), contains about 100,000compounds that are commercially available and also can be searched toidentify potential peptidomimetics of the alpha-amylase polypeptides.

In certain embodiments, an alpha-amylase polypeptide may furthercomprise post-translational modifications. Exemplary post-translationalprotein modifications include phosphorylation, acetylation, methylation,ADP-ribosylation, ubiquitination, glycosylation, carbonylation,sumoylation, biotinylation or addition of a polypeptide side chain or ofa hydrophobic group. As a result, the modified alpha-amylasepolypeptides may contain non-amino acid elements, such as lipids, poly-or mono-saccharides, and phosphates. Effects of such non-amino acidelements on the functionality of an alpha-amylase polypeptide may betested for its biological activity, for example, alpha-1,4-glucosidicbonds hydrolytic activity and/or its ability to treat Lafora Disease. Incertain embodiments, the alpha-amylase polypeptide may further compriseone or more polypeptide portions that enhance one or more of in vivostability, in vivo half life, uptake/administration, and/orpurification. In other embodiments, the internalizing moiety comprisesan antibody or an antigen-binding fragment thereof.

In some embodiments, an alpha-amylase polypeptide is not N-glycosylatedor lacks one or more of the N-glycosylation groups present in a wildtypealpha-amylase polypeptide. For example, the alpha-amylase polypeptidefor use in the present disclosure may lack all N-glycosylation sites,relative to native alpha-amylase, or the alpha-amylase polypeptide foruse in the present disclosure may be under-glycosylated, relative tonative alpha-amylase. In some embodiments, the alpha-amylase polypeptidecomprises a modified amino acid sequence that is unable to beN-glycosylated at one or more N-glycosylation sites. In someembodiments, asparagine (Asn) of at least one predicted N-glycosylationsite (i.e., a consensus sequence represented by the amino acid sequenceAsn-Xaa-Ser or Asn-Xaa-Thr) in the alpha-amylase polypeptide issubstituted by another amino acid. The disclosure contemplates that anyone or more of the foregoing examples can be combined so that analpha-amylase polypeptide of the present disclosure lacks one or moreN-glycosylation sites, and thus is either not glycosylated or is underglycosylated relative to native alpha-amylase.

In some embodiments, an alpha-amylase polypeptide is not O-glycosylatedor lacks one or more of the O-glycosylation groups present in a wildtypealpha-amylase polypeptide. In some embodiments, the alpha-amylasepolypeptide comprises a modified amino acid sequence that is unable tobe O-glycosylated at one or more O-glycosylation sites. In someembodiments, serine or threonine at any one or more predictedO-glycosylation site in the alpha-amylase polypeptide sequence issubstituted or deleted. The disclosure contemplates that any one or moreof the foregoing examples can be combined so that an alpha-amylasepolypeptide of the present disclosure lacks one or more N-glycosylationand/or O-glycosylation sites, and thus is either not glycosylated or isunder glycosylated relative to native alpha-amylase.

In one specific embodiment of the present disclosure, an alpha-amylasepolypeptide may be modified with nonproteinaceous polymers. In onespecific embodiment, the polymer is polyethylene glycol (“PEG”),polypropylene glycol, or polyoxyalkylenes, in the manner as set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337. PEG is a well-known, water soluble polymer that iscommercially available or can be prepared by ring-opening polymerizationof ethylene glycol according to methods well known in the art (Sandlerand Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages138-161).

By the terms “biological activity”, “bioactivity” or “functional” ismeant the ability of the alpha-amylase polypeptide to carry out thefunctions associated with wildtype alpha-amylase polypeptides, forexample, alpha-1,4-glucosidic bond hydrolytic activity. The terms“biological activity”, “bioactivity”, and “functional” are usedinterchangeably herein. As used herein, “fragments” are understood toinclude bioactive fragments (also referred to as functional fragments)or bioactive variants that exhibit “bioactivity” as described herein.That is, bioactive fragments or variants of alpha-amylase exhibitbioactivity that can be measured and tested. For example, bioactivefragments/functional fragments or variants exhibit the same orsubstantially the same bioactivity as native (i.e., wild-type, ornormal) alpha-amylase polypeptide, and such bioactivity can be assessedby the ability of the fragment or variant to, e.g., hydrolyzealpha-1,4-glucosidic bonds in a carbohydrate. As used herein,“substantially the same” refers to any parameter (e.g., activity) thatis at least 70% of a control against which the parameter is measured. Incertain embodiments, “substantially the same” also refers to anyparameter (e.g., activity) that is at least 75%, 80%, 85%, 90%, 92%,95%, 97%, 98%, 99%, 100%, 102%, 105%, or 110% of a control against whichthe parameter is measured. In certain embodiments, fragments or variantsof the alpha-amylase polypeptide will preferably retain at least 50%,60%, 70%, 80%, 85%, 90%, 95% or 100% of the alpha-amylase biologicalactivity associated with the native alpha-amylase polypeptide, whenassessed under the same or substantially the same conditions.

In certain embodiments, fragments or variants of the alpha-amylasepolypeptide have a half-life (t_(1/2)) which is enhanced relative to thehalf-life of the native protein. Preferably, the half-life ofalpha-amylase fragments or variants is enhanced by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%,300%, 400% or 500%, or even by 1000% relative to the half-life of thenative alpha-amylase polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf life, such as the half-life of the protein in the serum or otherbodily fluid of an animal. In addition, fragments or variants can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments or variants can be produced (recombinantly or by chemicalsynthesis) and tested to identify those fragments or variants that canfunction as well as or substantially similarly to a native alpha-amylasepolypeptide.

With respect to methods of increasing alpha-amylase bioactivity incells, the disclosure contemplates all combinations of any of theforegoing aspects and embodiments, as well as combinations with any ofthe embodiments set forth in the detailed description and examples. Thedescribed methods based on administering chimeric polypeptides orcontacting cells with chimeric polypeptides can be performed in vitro(e.g., in cells or culture) or in vivo (e.g., in a patient or animalmodel). In certain embodiments, the method is an in vitro method. Incertain embodiments, the method is an in vivo method.

In some aspects, the present disclosure also provides a method ofproducing any of the foregoing chimeric polypeptides as describedherein. Further, the present disclosure contemplates any number ofcombinations of the foregoing methods and compositions.

In certain aspects, an alpha-amylase polypeptide may be a fusion proteinwhich further comprises one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, and an immunoglobulin heavy chain constant region (Fc),maltose binding protein (MBP), which are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used. Fusion domains also include “epitope tags,”which are usually short peptide sequences for which a specific antibodyis available. Well known epitope tags for which specific monoclonalantibodies are readily available include FLAG, influenza virushaemagglutinin (HA), His and c-myc tags. An exemplary His tag has thesequence HHHHHH (SEQ ID NO: 7), and an exemplary c-myc tag has thesequence EQKLISEEDL (SEQ ID NO: 8). In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain embodiments, the alpha-amylasepolypeptides may contain one or more modifications that are capable ofstabilizing the polypeptides. For example, such modifications enhancethe in vitro half life of the polypeptides, enhance circulatory halflife of the polypeptides or reduce proteolytic degradation of thepolypeptides.

In certain embodiments of any of the foregoing, the alpha-amylaseportion of the chimeric polypeptide of the disclosure comprises analpha-amylase polypeptide, which in certain embodiments may be afunctional fragment of an alpha-amylase polypeptide or may be asubstantially full length alpha-amylase polypeptide. In someembodiments, the alpha-amylase polypeptide lacks the methionine at theN-terminal-most amino acid position (e.g., lacks the methionine at thefirst amino acid of any one of SEQ ID NOs: 44 or 45). Suitablealpha-amylase polypeptides for use in the chimeric polypeptides andmethods of the disclosure have alpha-1,4-glucosidic bond hydrolyticactivity, as evaluated in vitro or in vivo. Exemplary functionalfragments comprise, at least 100, 125, 150, 175, 200, 225, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, or 511 consecutive aminoacid residues of a full length alpha-amylase polypeptide (e.g., SEQ IDNOs: 44 or 45). In some embodiments, the functional fragment comprises100-150, 100-200, 100-250, 100-300, 100-400, 100-500, 100-511, 200-500,300-500, 400-500, 450-500, 475-500 or 500-511 consecutive amino acids ofa full-length alpha-amylase polypeptide (e.g., SEQ ID NO: 44 or 45).Similarly, in certain embodiments, the disclosure contemplates chimericproteins where the alpha-amylase portion is a variant of any of theforegoing alpha-amylase polypeptides or bioactive fragments. Exemplaryvariants have an amino acid sequence at least 90%, 92%, 95%, 96%, 97%,98%, or at least 99% identical to the amino acid sequence of a nativealpha-amylase polypeptide or functional fragment thereof, and suchvariants retain the alpha-amylase variant's alpha-1,4-glucosidic bondhydrolytic activity. The disclosure contemplates chimeric polypeptidesand the use of such polypeptides wherein the alpha-amylase portioncomprises any of the alpha-amylase polypeptides, fragments, or variantsdescribed herein in combination with any internalizing moiety describedherein. Moreover, in certain embodiments, the alpha-amylase portion ofany of the foregoing chimeric polypeptides may, in certain embodiments,by a fusion protein. Any such chimeric polypeptides comprising anycombination of alpha-amylase portions and internalizing moiety portions,and optionally including one or more linkers, one or more tags, etc.,may be used in any of the methods of the disclosure.

VI. Internalizing Moieties

The chimeric polypeptides for use in the methods disclosed hereincomprise an internalizing moiety. As used herein, the term“internalizing moiety” refers to a moiety capable of interacting with atarget tissue or a cell type to effect delivery of the attached moleculeinto the cell (i.e., penetrate desired cell; transport across a cellularmembrane; deliver across cellular membranes to, at least, thecytoplasm). Preferably, this disclosure relates to an internalizingmoiety which promotes delivery to, for example, muscle cells and livercells. Internalizing moieties having limited cross-reactivity aregenerally preferred. In certain embodiments, this disclosure relates toan internalizing moiety which selectively, although not necessarilyexclusively, targets and penetrates muscle cells. In certainembodiments, the internalizing moiety has limited cross-reactivity, andthus preferentially targets a particular cell or tissue type. However,it should be understood that internalizing moieties of the subjectdisclosure do not exclusively target specific cell types. Rather, theinternalizing moieties promote delivery to one or more particular celltypes, preferentially over other cell types, and thus provide fordelivery that is not ubiquitous. In certain embodiments, suitableinternalizing moieties include, for example, antibodies, monoclonalantibodies, or derivatives or analogs thereof. Other internalizingmoieties include for example, homing peptides, fusion proteins,receptors, ligands, aptamers, peptidomimetics, and any member of aspecific binding pair. In certain embodiments, the internalizing moietymediates transit across cellular membranes via an ENT2 transporter. Insome embodiments, the internalizing moiety helps the chimericpolypeptide effectively and efficiently transit cellular membranes. Insome embodiments, the internalizing moiety transits cellular membranesvia an equilibrative nucleoside (ENT) transporter. In some embodiments,the internalizing moiety transits cellular membranes via an ENT1, ENT2,ENT3 or ENT4 transporter. In some embodiments, the internalizing moietytransits or can transit cellular membranes via an equilibrativenucleoside transporter 2 (ENT2) and/or ENT3 transporter. In someembodiments, the internalizing moiety promotes delivery into musclecells (e.g., skeletal or cardiac muscle). In other embodiments, theinternalizing moiety promotes delivery into cells other than musclecells, e.g., neurons, epithelial cells, liver cells (e.g., hepatocytes),kidney cells or Leydig cells. For any of the foregoing, in certainembodiments, the internalizing moiety promotes delivery of a chimericpolypeptide into the cytoplasm.

In certain embodiments, the internalizing moiety is an antibody orantibody fragment that binds DNA. In other words, in certainembodiments, the antibody or antibody fragment (e.g., antibody fragmentcomprising an antigen binding fragment) binds DNA. In certainembodiments, DNA binding ability is measured versus a double strandedDNA substrate. In certain embodiments, the internalizing moiety is anantibody or antibody fragment that binds DNA and can transit cellularmembranes via ENT2.

In certain embodiments, the internalizing moiety promotes delivery of achimeric polypeptide into the cytoplasm. Without being bound by theory,regardless of whether the non-internalizing moiety polypeptide portionof the chimeric polypeptide comprises or consists of GAA, laforin,alpha-amylase, malin and/or AGL, its association with the internalizingmoiety portion facilitates delivery of the chimeric polypeptide, andthus, the non-internalizing moiety portion to the cytoplasm and,optionally, to the lysosome and/or autophagic vesicles. In certainembodiments, the internalizing moiety delivers GAA, laforin,alpha-amylase, malin and/or AGL activity into cells. In certainembodiments, the chimeric polypeptide of the disclosure comprises aGAA-containing chimeric polypeptide (e.g., the non-internalizing moietyportion comprises or consists of a GAA polypeptide). In certainembodiments, the chimeric polypeptide of the disclosure comprises anAGL-containing chimeric polypeptide (e.g., the non-internalizing moietyportion comprises or consists of an AGL polypeptide). In certainembodiments, the chimeric polypeptide of the disclosure comprises alaforin-containing chimeric polypeptide (e.g., the non-internalizingmoiety portion comprises or consists of a laforin polypeptide). Incertain embodiments, the chimeric polypeptide of the disclosurecomprises a malin-containing chimeric polypeptide (e.g., thenon-internalizing moiety portion comprises or consists of a malinpolypeptide). In certain embodiments, the chimeric polypeptide of thedisclosure comprises an alpha-amylase-containing chimeric polypeptide(e.g., the non-internalizing moiety portion comprises or consists of analpha-amylase polypeptide). Any of the internalizing moieties describedherein may be combined with any of the non-internalizing moietypolypeptide portions, as described herein, to generate a chimericpolypeptide of the disclosure.

In certain embodiments, the internalizing moiety is capable of bindingpolynucleotides. In certain embodiments, the internalizing moiety iscapable of binding DNA. In certain embodiments, the internalizing moietyis an antibody capable of binding DNA. In certain embodiments, theinternalizing moiety is capable of binding DNA with a K_(D) of less than1 In certain embodiments, the internalizing moiety is capable of bindingDNA with a K_(D) of less than 100 nM, less than 75 nM, less than 50 nM,or even less than 30 nM. K_(D) can be measured using Surface PlasmonResonance (SPR) or Quartz Crystal Microbalance (QCM), in accordance withcurrently standard methods. By way of example, a 3E10 antibody orantibody fragment, including an antibody or antibody fragment comprisinga VH having the amino acid sequence set forth in SEQ ID NO: 9 and a VLhaving an amino acid sequence set forth in SEQ ID NO: 10 is known tobind DNA with a K_(D) of less than 100 nM. Thus, in certain embodiments,an internalizing moiety for use in the chimeric polypeptides of thedisclosure is an antibody or antibody fragment (e.g., an antigen bindingfragment) that can transit cellular membranes into the cytoplasm andbinds to DNA. This is also exemplary of an anti-DNA antibody. In certainembodiments, an internalizing moiety for use herein is an anti-DNAantibody or antigen binding fragment thereof.

In fact, a full length antibody comprising the foregoing VH and VL bindsa double-stranded blunt DNA substrate with an even lower K_(D), asevaluated by ELISA. In certain embodiments, the internalizing moietybinds double-stranded, blunt DNA, and DNA binding activity is or can bedemonstrated in a binding assay using blunt DNA (see, for example, Xuet. Al. (2009) EMBO Journal 28: 568-577; Hansen et al., (2012) SciTranslation Med 4: DOI 10.1126/scitranslmed.3004385), such as by ELISA,QCM, or Biacore. In certain embodiments, the foregoing K_(D) of theantibody or antibody fragment (such as an antibody fragment comprisingan antigen-binding fragment) is evaluated versus a double stranded,blunt end DNA substrate, such as the DNA substrate set forth in Xu etal. (e.g., a DNA comprising two strands, wherein one of the strandsconsists of the following sequence: 5′ - GGG TGA ACC TGC AGG TGG GCA AAGATG TCC-3′). In certain embodiments, the internalizing moiety is ananti-DNA antibody. In certain embodiments, the internalizing moiety is aFab, a Fab′, or a full length antibody. It is recognized that 3E10 andother anti-DNA antibodies may be capable of binding a variety of DNAsubstrates with high affinity, as has been demonstrated.

In some embodiments, the internalizing moiety targets a GAA polypeptide(e.g., mature GAA polypeptide), laforin polypeptide, alpha-amylasepolypeptide, malin polypeptide and/or AGL polypeptide to muscle cells,and mediates transit of the polypeptide across the cellular membraneinto the cytoplasm of the muscle cells. In some embodiments, theinternalizing moiety targets a GAA polypeptide (e.g., mature GAApolypeptide), laforin polypeptide, alpha-amylase polypeptide, malinpolypeptide and/or AGL polypeptide to liver or neuronal cells, andmediates transit of the polypeptide across the cellular membrane intothe cytoplasm of the liver or neuronal cells.

As used herein, the term “internalizing moiety” refers to a moietycapable of interacting with a target tissue or a cell type. Preferably,this disclosure relates to an internalizing moiety which promotesdelivery to, for example, muscle cells and liver cells. Internalizingmoieties having limited cross-reactivity are generally preferred.However, it should be understood that internalizing moieties of thesubject disclosure do not exclusively target specific cell types.Rather, the internalizing moieties generally, in certain embodiments,promote delivery to one or more particular cell types, preferentiallyover other cell types, and thus provide for delivery that is notubiquitous. In certain embodiments, suitable internalizing moietiesinclude, for example, antibodies, monoclonal antibodies, or derivativesor analogs thereof; and other internalizing moieties include forexample, homing peptides, fusion proteins, receptors, ligands, aptamers,peptidomimetics, and any member of a specific binding pair. In someembodiments, the internalizing moiety helps the chimeric polypeptideeffectively and efficiently transit cellular membranes. In someembodiments, the internalizing moiety transits cellular membranes via anequilibrative nucleoside (ENT) transporter. In some embodiments, theinternalizing moiety transits cellular membranes via an ENT1, ENT2, ENT3or ENT4 transporter. In some embodiments, the internalizing moietytransits or can transit cellular membranes via an equilibrativenucleoside transporter 2 (ENT2) and/or ENT3 transporter. In someembodiments, the internalizing moiety promotes delivery into musclecells (e.g., skeletal or cardiac muscle). In other embodiments, theinternalizing moiety promotes delivery into cells other than musclecells, e.g., neurons, epithelial cells, liver cells, kidney cells orLeydig cells. In certain embodiments, the internalizing moiety promotesdelivery into, at least, muscle cells and liver cells.

(a) Antibodies

In certain aspects, an internalizing moiety may comprise an antibody,including a monoclonal antibody, a polyclonal antibody, and a humanizedantibody. Without being bound by theory, such antibody may bind to anantigen of a target tissue and thus mediate the delivery of the subjectchimeric polypeptide to the target tissue (e.g., muscle, neuronal and/orliver cells). In some embodiments, internalizing moieties may compriseantibody fragments, derivatives or analogs thereof, including withoutlimitation: antibody fragments comprising antigen binding fragments(e.g., Fv fragments, single chain Fv (scFv) fragments, Fab′ fragments,F(ab′)2 fragments), single domain antibodies, camelized antibodies andantibody fragments, humanized antibodies and antibody fragments, humanantibodies and antibody fragments, and multivalent versions of theforegoing; multivalent internalizing moieties including withoutlimitation: Fv fragments, single chain Fv (scFv) fragments, Fab′fragments, F(ab′)2 fragments, single domain antibodies, camelizedantibodies and antibody fragments, humanized antibodies and antibodyfragments, human antibodies and antibody fragments, and multivalentversions of the foregoing; multivalent internalizing moieties includingwithout limitation: monospecific or bispecific antibodies, such asdisulfide stabilized Fv fragments, scFv tandems ((scFv)₂ fragments),diabodies, tribodies or tetrabodies, which typically are covalentlylinked or otherwise stabilized (i.e., leucine zipper or helixstabilized) scFv fragments; receptor molecules which naturally interactwith a desired target molecule. In some embodiments, the antibodies orvariants thereof may be chimeric, e.g., they may include variable heavyor light regions from the murine 3E10 antibody, but may include constantregions from an antibody of another species (e.g., a human). In someembodiments, the antibodies or variants thereof may comprise a constantregion that is a hybrid of several different antibody subclass constantdomains (e.g., any combination of IgG1, IgG2a, IgG2b, IgG3 and IgG4,from any species or combination of species). In some embodiments, theantibodies or variants thereof (e.g., the internalizing moiety portionof the chimeric polypeptide) comprise the following constant domainscheme: IgG2a CH1-IgG1 hinge-IgG1 CH2-CH3, for example, any of theforegoing may be human IgG or murine IgG. Other suitable combinationsare also contemplated. In other embodiments, the antibody comprises afull length antibody and the CH1, hinge, CH2, and CH3 is from the sameconstant domain subclass (e.g., IgG1). In some embodiments, theantibodies or variants thereof are antibody fragments (e.g., theinternalizing moiety is an antibody fragment comprising an antigenbinding fragment; e.g., the internalizing moiety is an antigen bindingfragment) comprising a portion of the constant domain of animmunoglobulin, for example, the following constant domain scheme: IgG2aCH1-IgG1 upper hinge. In some embodiments, the antibodies or variantsthereof comprise a kappa constant domain (e.g., SEQ ID NO: 34). Heavychain constant domains (whether for a full length antibody or for anantibody fragment (e.g., an antigen binding fragment) comprising anamino acid substitution, relative to native IgG domains, to decreaseeffector function and/or facilitate production are included within thescope of antibodies and antigen binding fragments. For example, one,two, three, or four amino acid substitutions in a heavy chain, relativeto a native murine or human immunoglobulin constant region, such as inthe hinge or CH2 domain of a heavy chain constant region.

In certain embodiments, internalizing moiety comprises an antibody, andthe heavy chain comprises a VH region, and a constant domain comprisinga CH1, hinge, CH2, and CH3 domain. In certain embodiments, a heavy chaincomprises a VH region, and a constant domain comprising a CH1 domainand, optionally, the upper hinge. The upper hinge may include, forexample, 1, 2, 3, or 4 amino acid residues of the hinge region. Incertain embodiments, the upper hinge does not include a cysteineresidue. In certain embodiments, the upper hinge includes one or moreconsecutive residues N-terminal to a cysteine that exists in the nativehinge sequence. In certain embodiments, the heavy chain comprises a CHregion, and a constant domain comprising a CH1 domain and a hinge. Incertain embodiments, the hinge (whether present as part of a full lengthantibody or an antibody fragment) comprises a C to S substitution at aposition corresponding to Kabat position 222 (e.g., a C222S in thehinge, where the variation is at a position corresponding to Kabatposition 222). In other words, in certain embodiments, the internalizingmoiety comprises a serine residue, rather than a cysteine residue, in ahinge domain at a position corresponding to Kabat 222. In certainembodiments, the heavy chain comprises a constant domain comprising aCH1, hinge, CH2 and, optionally CH3 domain. In certain embodiments, aCH2 domain comprises an N to Q substitution at a position correspondingto Kabat position 297 (e.g., a N297Q in a CH2 domain, wherein thevariation is at a position corresponding to Kabat position 297). Inother words, in certain embodiments, the internalizing moiety comprisesa glutamine, rather than an asparagine, at a position corresponding toKabat position 297.

In some embodiments, the internalizing moiety comprises all or a portionof the Fc region of an immunoglobulin. In other words, in addition to anantigen binding portion, in certain embodiments, the internalizingmoiety comprises all or a portion of a heavy chain constant region of animmunoglobulin (e.g., one or two polypeptide chains of a heavy chainconstant region. As is known, each immunoglobulin heavy chain constantregion comprises four or five domains. The domains are namedsequentially as follows: CH1-hinge-CH2-CH3(-CH4). The DNA sequences ofthe heavy chain domains have cross-homology among the immunoglobulinclasses, e.g., the CH2 domain of IgG is homologous to the CH2 domain ofIgA and IgD, and to the CH3 domain of IgM and IgE. As used herein, theterm, “immunoglobulin Fc region” is understood to mean thecarboxyl-terminal portion of an immunoglobulin chain constant region,preferably an immunoglobulin heavy chain constant region, or a portionthereof. For example, an immunoglobulin Fc region may comprise 1) a CH1domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3domain, or 5) a combination of two or more domains and an immunoglobulinhinge region, or a portion of a hinger (e.g., an upper hinge). Incertain embodiments, an internalizing moiety further comprises a lightchain constant region (CL).

In one embodiment, the class of immunoglobulin from which the heavychain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or4). Other classes of immunoglobulin, IgA (Igα), IgD (Igδ), IgE (Igß) andIgM (Igμ), may be used. The choice of particular immunoglobulin heavychain constant region sequences from certain immunoglobulin classes andsubclasses to achieve a particular result is considered to be within thelevel of skill in the art. The portion of the DNA construct encoding theimmunoglobulin Fc region preferably comprises at least a portion of ahinge domain, and preferably at least a portion of a CH3 domain of Fc γor the homologous domains in any of IgA, IgD, IgE, or IgM. Furthermore,it is contemplated that substitution or deletion of amino acids withinthe immunoglobulin heavy chain constant regions may be useful in thepractice of the disclosure. One example would be to introduce amino acidsubstitutions in the upper CH2 region to create a Fc variant withreduced affinity for Fc receptors (Cole et al. (1997) J. IMMUNOL.159:3613). One of ordinary skill in the art can prepare such constructsusing well known molecular biology techniques.

In certain embodiments, the antibodies or variants thereof, may bemodified to make them less immunogenic when administered to a subject.For example, if the subject is human, the antibody may be “humanized”;where the complementarity determining region(s) of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones, P. et al. (1986), Nature, 321, 522-525 orTempest et al. (1991), Biotechnology, 9, 266-273. The term humanizationand humanized is well understood in the art when referring toantibodies. In some embodiments, the internalizing moiety is any peptideor antibody-like protein having the complementarity determining regions(CDRs) of the 3E10 antibody sequence, or of an antibody that binds thesame epitope (e.g., the same target, such as DNA) as 3E10. Also,transgenic mice, or other mammals, may be used to express humanized orhuman antibodies. Such humanization may be partial or complete.

In certain embodiments, the internalizing moiety comprises themonoclonal antibody 3E10 or an antigen binding fragment thereof. Inother embodiments, the internalizing moiety comprises an antibody or anantigen binding fragment thereof, such as any of the antigen bindingfragments described herein. For example, the antibody or antigen bindingfragment thereof may be monoclonal antibody 3E10, or a variant thereofthat retains cell penetrating activity, or an antigen binding fragmentof 3E10 or said 3E10 variant. Additionally, the antibody or antigenbinding fragment thereof may be an antibody that binds to the sameepitope (e.g., target, such as DNA) as 3E10, or an antibody that hassubstantially the same cell penetrating activity as 3E10, or an antigenbinding fragment thereof. These are exemplary of agents that can transitcells via ENT2. In certain embodiments, the internalizing moiety iscapable of binding polynucleotides. In certain embodiments, theinternalizing moiety is capable of binding DNA, such as double-strandedblunt DNA. In certain embodiments, the internalizing moiety is capableof binding DNA with a K_(D) of less than 100 nM. In certain embodiments,the internalizing moiety is capable of binding DNA with a K_(D) of lessthan 100 nM, less than 75 nM, less than 50 nM, or even less than 30 nM.K_(D) is determined using SPR or QCM or ELISA, according tomanufacturer's instructions and current practice. In certainembodiments, K_(D), with respect to binding to double stranded blunt DNAis evaluated using the following DNA as substrate: is evaluated versus adouble stranded, blunt end DNA substrate, such as the DNA substrate setforth in Xu et al. (e.g., a DNA comprising two strands, wherein one ofthe strands consists of the following sequence: 5′ - GGG TGA ACC TGC AGGTGG GCA AAG ATG TCC-3′. In certain embodiments, the internalizing moietyis an anti-DNA antibody or antigen binding fragment.

In certain embodiments, the antigen binding fragment is an Fv or scFvfragment thereof. Monoclonal antibody 3E10 can be produced by ahybridoma 3E10 placed permanently on deposit with the American TypeCulture Collection (ATCC) under ATCC accession number PTA-2439 and isdisclosed in U.S. Pat. No. 7,189,396. This antibody has been shown tobind DNA. Additionally or alternatively, the 3E10 antibody can beproduced by expressing in a host cell nucleotide sequences encoding theheavy and light chains of the 3E10 antibody. The term “3E10 antibody” or“monoclonal antibody 3E10” are used to refer to the antibody, regardlessof the method used to produce the antibody. Similarly, when referring tovariants or antigen-binding fragments of 3E10, such terms are usedwithout reference to the manner in which the antibody was produced. Atthis point, 3E10 is generally not produced by the hybridoma but isproduced recombinantly. Thus, in the context of the present application,3E10 antibody, unless otherwise specified, will refer to an antibodyhaving the sequence of the hybridoma or comprising a variable heavychain domain comprising the amino acid sequence set forth in SEQ ID NO:9 (which has a one amino acid substitution relative to that of the 3E10antibody deposited with the ATCC, as described herein) and the variablelight chain domain comprising the amino acid sequence set forth in SEQID NO: 10, and antibody fragments thereof.

The internalizing moiety may also comprise variants of mAb 3E10, such asvariants of 3E10 which retain the same cell penetration characteristicsas mAb 3E10, as well as variants modified by mutation to improve theutility thereof (e.g., improved ability to target specific cell types,improved ability to penetrate the cell membrane, improved ability tolocalize to the cellular DNA, convenient site for conjugation, and thelike). Such variants include variants wherein one or more conservativesubstitutions are introduced into the heavy chain, the light chainand/or the constant region(s) of the antibody. Such variants includehumanized versions of 3E10 or a 3E10 variant. In some embodiments, thelight chain or heavy chain may be modified at the N-terminus orC-terminus. Similarly, the foregoing description of variants applies toantigen binding fragments. Any of these antibodies, variants, orfragments may be made recombinantly by expression of the nucleotidesequence(s) in a host cell.

Monoclonal antibody 3E10 has been shown to penetrate cells to deliverproteins and nucleic acids into the cytoplasmic or nuclear spaces oftarget tissues (Weisbart R H et al., J Autoimmun. 1998 Oct;11(5):539-46;Weisbart R H, et al. Mol Immunol. 2003 March; 39(13):783-9; Zack DJ etal., J Immunol. 1996 Sep 1;157(5):2082-8.). Further, the VH and Vksequences of 3E10 are highly homologous to human antibodies, withrespective humanness z-scores of 0.943 and -0.880. Thus, Fv3E10 isexpected to induce less of an anti-antibody response than many otherapproved humanized antibodies (Abhinandan K R et al., Mol. Biol. 2007369, 852-862). A single chain Fv fragment of 3E10 possesses all the cellpenetrating capabilities of the original monoclonal antibody, andproteins such as catalase, dystrophin, HSP70 and p53 retain theiractivity following conjugation to Fv3E10 (Hansen J E et al., Brain Res.2006 May 9; 1088(1):187-96; Weisbart R H et al., Cancer Lett. 2003 Jun.10; 195(2):211-9; Weisbart R H et al., J Drug Target. 2005 February;13(2):81-7; Weisbart R H et al., J Immunol. 2000 Jun. 1; 164(11):6020-6;Hansen J E et al., J Biol Chem. 2007 Jul. 20; 282(29):20790-3). The 3E10is built on the antibody scaffold present in all mammals; a mousevariable heavy chain and variable kappa light chain. 3E10 can gain entryto cells via the ENT2 nucleotide transporter that is particularlyenriched in skeletal muscle and cancer cells, and in vitro studies haveshown that 3E10 is nontoxic. (Weisbart R H et al., Mol Immunol. 2003March; 39(13):783-9; Pennycooke M et al., Biochem Biophys Res Commun.2001 Jan. 26; 280(3):951-9). 3E10 may also be capable of transitingmembranes via ENT3.

The internalizing moiety may also include mutants of mAb 3E10, such asvariants of 3E10 which retain the same or substantially the same cellpenetration characteristics as mAb 3E10, as well as variants modified bymutation to improve the utility thereof (e.g., improved ability totarget specific cell types, improved ability to penetrate the cellmembrane, improved ability to localize to the cellular DNA, improvedbinding affinity, and the like). Such mutants include variants whereinone or more conservative substitutions are introduced into the heavychain, the light chain and/or the constant region(s) of the antibody.Numerous variants of mAb 3E10 have been characterized in, e.g., U.S.Pat. No. 7,189,396 and WO 2008/091911, the teachings of which areincorporated by reference herein in their entirety.

In certain embodiments, the internalizing moiety comprises an antibodyor antigen binding fragment comprising an VH domain comprising an aminoacid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 99%, or 100%identical to SEQ ID NO: 9 and/or a VL domain comprising an amino acidsequence at least 85%, 90%, 95%, 96%, 97%, 99%, or 100% identical to SEQID NO: 10, or a humanized variant thereof. In some embodiments, theinternalizing moiety comprises any of the light chain variable domaindescribed herein and a kappa constant domain (CL) having an amino acidsequence at least 85%, 90%, 95%, 96%, 97%, 99%, or 100% identical to SEQID NO: 34. In some embodiments, the internalizing moiety comprises anamino acid sequence at least 85%, 90%, 95%, 96%, 97%, 99%, or 100%identical to SEQ ID NO: 35. In some embodiments, the internalizingmoiety comprises an amino acid sequence at least 85%, 90%, 95%, 96%,97%, 99%, or 100% identical to SEQ ID NO: 37. It is understood that,when a signal sequence is included for expression of an antibody orantibody fragment, that signal sequence is generally cleaved and notpresented in the finished chimeric polypeptide (e.g., the signalsequence is generally cleaved and present only transiently duringprotein production). Such internalizing moieties can transit, in certainembodiments, cells via ENT2 and/or bind DNA.

In certain embodiments, the internalizing moiety is capable of bindingpolynucleotides. In certain embodiments, the internalizing moiety iscapable of binding (specifically binding) DNA. In certain embodiments,the internalizing moiety is capable of binding DNA with a K_(D) of lessthan 100 nM. In certain embodiments, the internalizing moiety is capableof binding DNA with a K_(D) of less than 50 nM. In certain embodiments,the internalizing moiety is an anti-DNA antibody, such as an antibody orantigen binding fragment that binds double-stranded blunt DNA. Incertain embodiments, the internalizing moiety is an anti-DNA antibody orantigen binding fragment (thereof), where K_(D) is evaluated versus adouble stranded DNA substrate, such as provided herein.

In certain embodiments, the internalizing moiety is an antigen bindingfragment, such as a single chain Fv of 3E10 (scFv) comprising SEQ IDNOs: 9 and 10. In certain embodiments, the internalizing moietycomprises a single chain Fv of 3E10 (or another antigen bindingfragment), and the amino acid sequence of the V_(H) domain is at least90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9, andamino acid sequence of the V_(L) domain is at least 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 10. The variant 3E10 orfragment thereof retains the function of an internalizing moiety. Whenthe internalizing moiety is an scFv, the VH and VL domains are typicallyconnected via a linker, such as a gly/ser linker. The VH domain may beN-terminal to the VL domain or vice versa.

In certain embodiments, the internalizing moiety is an antigen bindingfragment, such as a Fab comprising a VH and a VL. In certainembodiments, the internalizing moiety is a Fab (or another antigenbinding fragment, such as a Fab′), and the amino acid sequence of theV_(H) domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 9. In certain embodiments, the internalizing moiety is aFab (or another antigen binding fragment, such as a Fab′), and the aminoacid sequence of the V_(L) domain is at least 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 10. Our VH and VL domains, orcombinations thereof, described herein are similarly contemplated. Incertain embodiments, when the internalizing moiety is a Fab the heavychain comprises a CH1 domain and an upper hinge of an immunoglobulinconstant region. In certain embodiments, the upper hinge comprises asubstitution, relative to a native immunoglobulin constant region, suchas to decrease effector function and/or to eliminate a cysteine (e.g., aC to S). In certain embodiments, the upper hinge does not include acysteine.

In certain embodiments, the constant domain of the antibody or antibodyfragment (e.g., antigen binding fragment) comprises all or a portion ofa human Fc domain. In certain embodiments, the internalizing moiety is afull length antibody, and the constant domain of the antibody comprisesa CH1, hinge, CH2 and CH3 domain. In certain embodiments, the constantdomain comprises one or more substitutions, relative to a nativeimmunoglobulin, that reduce effector function. Optionally, in certainembodiments, such a constant domain may include one or more (e.g., 1substitution, 2 substitutions, 3 substitutions) substitutions in theheavy chain constant domain, such as in the hinge and/or CH2 domains,such as to reduce effector function. Such substitutions are known in theart.

In certain embodiments, the internalizing moiety is an antigen bindingfragment - a fragment of an antibody comprising an antigen bindingfragment. Suitable such fragments of antibodies, such as scFv, Fab, Fab′and the like are described herein. In certain embodiments, theinternalizing moiety is an antigen binding fragment or a full lengthantibody. In certain embodiments, the internalizing moiety comprises alight chain comprising a constant region (CL). In certain embodiments,the internalizing moiety comprises a heavy chain comprising a constantregion, wherein the constant region comprises a CH1 domain. In certainembodiments, the internalizing moiety comprises a heavy chain comprisinga constant region and a light chain comprising a constant region,wherein the heavy chain constant region comprises a CH1 domain.Optionally, the internalizing moiety may further comprise a heavy chainconstant region comprising all or a portion of a hinge (e.g., an upperhinge or more than the upper hinge). Optionally, the internalizingmoiety may further comprise a heavy chain comprising a CH2 and/or CH3domain.

In some embodiments, the internalizing moiety comprises one or more ofthe CDRs of the 3E10 antibody. In certain embodiments, the internalizingmoiety comprises one or more of the CDRs of a 3E10 antibody comprisingthe amino acid sequence of a V_(H) domain that is identical to SEQ IDNO: 9 and the amino acid sequence of a V_(L) domain that is identical toSEQ ID NO: 10. The CDRs of the 3E10 antibody may be determined using anyof the CDR identification schemes available in the art. For example, insome embodiments, the CDRs of the 3E10 antibody are defined according tothe Kabat definition as set forth in Kabat et al. Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991). In other embodiments, theCDRs of the 3E10 antibody are defined according to Chothia et al., 1987,J Mol Biol. 196: 901-917 and Chothia et al., 1989, Nature. 342:877-883.In other embodiments, the CDRs of the 3E10 antibody are definedaccording to the international ImMunoGeneTics database (IMGT) as setforth in LeFranc et al., 2003, Development and Comparative Immunology,27: 55-77. In other embodiments, the CDRs of the 3E10 antibody aredefined according to Honegger A, Pluckthun A., 2001, J Mol Biol.,309:657-670. In some embodiments, the CDRs of the 3E10 antibody aredefined according to any of the CDR identification schemes discussed inKunik et al., 2012, PLoS Comput Biol. 8(2): e1002388. In order to numberresidues of a 3E10 antibody for the purpose of identifying CDRsaccording to any of the CDR identification schemes known in the art, onemay align the 3E10 antibody at regions of homology of the sequence ofthe antibody with a “standard” numbered sequence known in the art forthe elected CDR identification scheme. Maximal alignment of frameworkresidues frequently requires the insertion of “spacer” residues in thenumbering system, to be used for the Fv region. In addition, theidentity of certain individual residues at any given site number mayvary from antibody chain to antibody chain due to interspecies orallelic divergence.

In certain embodiments, the internalizing moiety comprises at least 1,2, 3, 4, or 5 of the CDRs of 3E10 as determined using the Kabat CDRidentification scheme (e.g., the CDRs set forth in SEQ ID NOs: 13-18;the internalizing moiety is an antibody or antigen binding fragmentthereof comprising a heavy chain comprising CDR1, CDR2, and CDR 3, asset forth in SEQ ID NOs: 13, 14, and 15, respectively, and a light chaincomprising CDR1, CDR2, and CDR3, as set forth in SEQ ID NOs: 16, 17 and18, respectively; e.g., and these CDRs in the internalizing moiety areas determined using the Kabat scheme). In certain embodiments, theantibody or antigen binding fragment comprises a VH CDR2 as set forth inSEQ ID NO: 46 and/or a VL CDR2 as set forth in SEQ ID NO: 48 and/or a VLCDR1 as set forth in SEQ ID NO: 47.

In certain embodiments, the antibodies and antigen binding fragments ofthe disclosure comprise a variable heavy chain domain comprising one orat least one CDR different from the corresponding CDR set forth in SEQID NO: 9, as determined using the Kabat CDR identification scheme. Insome embodiments, the one or at least one different CDR is V_(H) CDR2 asset forth in SEQ ID NO: 46.

In certain embodiments, the antibodies and antigen binding fragments ofthe disclosure comprise a variable light chain domain comprising one orat least one CDR different from the corresponding CDR set forth in SEQID NO: 10, as determined using the Kabat CDR identification scheme. Insome embodiments, the one or at least one different CDR is a V_(L) CDR1as set forth in SEQ ID NO: 47. In some embodiments, the one or at leastone different CDR is a V_(L) CDR2 as set forth in SEQ ID NO: 48.

In certain embodiments, the antibody or antigen binding fragmentcomprises a VH CDR2 as set forth in SEQ ID NO: 46 and/or a VL CDR2 asset forth in SEQ ID NO: 48 and/or a VL CDR1 as set forth in SEQ ID NO:47.

In other embodiments, the internalizing moiety comprises at least 1, 2,3, 4 or 5 of the CDRs of 3E10 as determined using the IMGTidentification scheme (e.g., the CDRs set forth in SEQ ID NOs: 24-29;the internalizing moiety is an antibody or antigen binding fragmentthereof comprising a heavy chain comprising CDR1, CDR2, and CDR 3, asset forth in SEQ ID NOs: 24, 25, and 26, respectively, and a light chaincomprising CDR1, CDR2, and CDR3, as set forth in SEQ ID NOs: 27, 28, and29, respectively; e.g., and these CDRs in the internalizing moiety areas determined using the IMGT identification scheme). In certainembodiments, the internalizing moiety comprises all six CDRs of 3E10 asdetermined using the Kabat CDR identification scheme (e.g., comprisesSEQ ID NOs 13-18). In other embodiments, the internalizing moietycomprises all six CDRS of 3E10 as determined using the IMGTidentification scheme (e.g., which are set forth as SEQ ID NOs: 24-29).

In certain embodiments, the antibody or antigen binding fragmentcomprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO 13;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 14;

a VH CDR3 having the amino acid sequence of SEQ ID NO: 15;

a VL CDR1 having the amino acid sequence of SEQ ID NO: 16;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 17; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 18; which CDRsare according to Kabat.

In certain embodiments, the antibody or antigen binding fragmentcomprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO: 13;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 46; and

a VH CDR3 having the amino acid sequence of SEQ ID NO: 15,

a VL CDR1 having the amino acid sequence of SEQ ID NO: 16 or 47;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 48; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 18, which CDRsare according to Kabat.

In certain embodiments, the antibody or antigen binding fragmentcomprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO: 13;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 46; and

a VH CDR3 having the amino acid sequence of SEQ ID NO: 15,

a VL CDR1 having the amino acid sequence of SEQ ID NO: 16;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 48; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 18, which CDRsare according to Kabat.

In certain embodiments, the antibody or antigen binding fragmentcomprises:

a VH CDR1 having the amino acid sequence of SEQ ID NO 24;

a VH CDR2 having the amino acid sequence of SEQ ID NO: 25;

a VH CDR3 having the amino acid sequence of SEQ ID NO: 26;

a VL CDR1 having the amino acid sequence of SEQ ID NO: 27;

a VL CDR2 having the amino acid sequence of SEQ ID NO: 28; and

a VL CDR3 having the amino acid sequence of SEQ ID NO: 29; which CDRsare according to the IMGT system.

For any of the foregoing, in certain embodiments, the internalizingmoiety is an antibody that binds the same epitope (e.g., the sametarget, such as DNA) as 3E10 and/or the internalizing moiety competeswith 3E10 for binding to antigen. Exemplary internalizing moieties cantarget and transit cells via ENT2. Exemplary internalizing moietiescomprise antibodies or antigen binding fragments that bind DNA, such asdouble stranded blunt DNA.

In certain embodiments, the internalizing moiety comprising an antibodyfragment, and the antibody fragment comprises an antigen bindingfragment, such as an Fab or Fab′. In other words, in certainembodiments, the internalizing moiety comprises an Fab or Fab′.

In certain embodiments, the internalizing moiety competes with bindingfor a DNA substrate, such as double-stranded blunt DNA, with an antibody(or antigen-binding fragment) of the antibody produced by hybridoma 3E10placed permanently on deposit with the American Type Culture Collection(ATCC) under ATCC accession number PTA-2439.

The present disclosure utilizes the cell penetrating ability of 3E10 or3E10 fragments or variants to promote delivery of GAA (e.g., mature GAAor GAA polypeptides comprising mature GAA), laforin, alpha-amylase,malin and/or AGL in vivo or into cells in vitro, such as into cytoplasmof cells. 3E10 and 3E10 variants and fragments are particularly wellsuited for this because of their demonstrated ability to effectivelypromote delivery to muscle cells, including skeletal and cardiac muscle,as well as diaphragm. Thus, in certain embodiments, 3E10 and 3E10variants and fragments (or antibodies or antibody fragments that bindthe same epitope and/or transit cells via ENT2) are useful for promotingeffective delivery into cells in subjects, such as human patients ormodel organisms, having Forbes-Cori and/or Andersen Disease and/or PompeDisease and/or von Gierke Disease and/or Lafora Disease or symptoms thatrecapitulate Forbes-Cori and/or Andersen Disease and/or Pompe Diseaseand/or von Gierke Disease and/or Lafora Disease. In certain embodiments,chimeric polypeptides in which the internalizing moiety is related to3E10 are suitable to facilitate delivery of a GAA polypeptide comprisingGAA ((e.g., mature GAA polypeptide), laforin, alpha-amylase, malinand/or AGL to the cytoplasm of cells.

As described further below, a recombinant 3E10 or 3E10-like variant orfragment can be conjugated, linked or otherwise joined to a GAApolypeptide, such as to a GAA polypeptide comprising a mature GAApolypeptide. In some embodiments, a recombinant 3E10 or 3E10-likevariant or fragment can be conjugated, linked or otherwise joined to alaforin polypeptide. In some embodiments, a recombinant 3E10 or3E10-like variant or fragment can be conjugated, linked or otherwisejoined to an AGL polypeptide. In some embodiments, a recombinant 3E10 or3E10-like variant or fragment can be conjugated, linked or otherwisejoined to a malin polypeptide. In some embodiments, a recombinant 3E10or 3E10-like variant or fragment can be conjugated, linked or otherwisejoined to an alpha-amylase polypeptide. In the context of makingchimeric polypeptides to GAA, laforin, alpha-amylase, malin and/or AGL,chemical conjugation, as well as making the chimeric polypeptide as afusion protein is available and known in the art.

Preparation of antibodies or fragments thereof (e.g., a single chain Fvfragment encoded by V_(H)-linker-V_(L) or V_(L)-linker-V_(H) or a Fab)is well known in the art. In particular, methods of recombinantproduction of mAb 3E10 antibody fragments have been described in WO2008/091911. Further, methods of generating scFv fragments of antibodiesor Fabs are well known in the art. When recombinantly producing anantibody or antibody fragment, a linker may be used. For example,typical surface amino acids in flexible protein regions include Gly, Asnand Ser. One exemplary linker is provided in SEQ ID NO: 5, 6 or 30.Permutations of amino acid sequences containing Gly, Asn and Ser wouldbe expected to satisfy the criteria (e.g., flexible with minimalhydrophobic or charged character) for a linker sequence. Anotherexemplary linker is of the formula (G4S)n, wherein n is an integer from1-10, such as 2, 3, or 4. Other near neutral amino acids, such as Thrand Ala, can also be used in the linker sequence.

In addition to linkers interconnecting portions of, for example, anscFv, the disclosure contemplates the use of additional linkers to, forexample, interconnect the GAA, laforin, alpha-amylase, malin and/or AGLportion to the antibody portion of the chimeric polypeptide.

Preparation of antibodies may be accomplished by any number ofwell-known methods for generating monoclonal antibodies. These methodstypically include the step of immunization of animals, typically mice,with a desired immunogen (e.g., a desired target molecule or fragmentthereof). Once the mice have been immunized, and preferably boosted oneor more times with the desired immunogen(s), monoclonalantibody-producing hybridomas may be prepared and screened according towell known methods (see, for example, Kuby, Janis, Immunology, ThirdEdition, pp. 131-139, W.H. Freeman & Co.

(1997), for a general overview of monoclonal antibody production, thatportion of which is incorporated herein by reference). Over the pastseveral decades, antibody production has become extremely robust. Invitro methods that combine antibody recognition and phage displaytechniques allow one to amplify and select antibodies with very specificbinding capabilities. See, for example, Holt, L. J. et al., “The Use ofRecombinant Antibodies in Proteomics,” Current Opinion in Biotechnology,2000, 11:445-449, incorporated herein by reference. These methodstypically are much less cumbersome than preparation of hybridomas bytraditional monoclonal antibody preparation methods. In one embodiment,phage display technology may be used to generate an internalizing moietyspecific for a desired target molecule. An immune response to a selectedimmunogen is elicited in an animal (such as a mouse, rabbit, goat orother animal) and the response is boosted to expand theimmunogen-specific B-cell population. Messenger RNA is isolated fromthose B-cells, or optionally a monoclonal or polyclonal hybridomapopulation. The mRNA is reverse-transcribed by known methods usingeither a poly-A primer or murine immunoglobulin-specific primer(s),typically specific to sequences adjacent to the desired VH and V_(L)chains, to yield cDNA. The desired VH and V_(L) chains are amplified bypolymerase chain reaction (PCR) typically using VH and V_(L) specificprimer sets, and are ligated together, separated by a linker. VH andV_(L) specific primer sets are commercially available, for instance fromStratagene, Inc. of La Jolla, Calif. Assembled V_(H)-linker-V_(L)product (encoding an scFv fragment) is selected for and amplified byPCR. Restriction sites are introduced into the ends of theV_(H)-linker-V_(L) product by PCR with primers including restrictionsites and the scFv fragment is inserted into a suitable expressionvector (typically a plasmid) for phage display. Other fragments, such asan Fab′ fragment, may be cloned into phage display vectors for surfaceexpression on phage particles. The phage may be any phage, such aslambda, but typically is a filamentous phage, such as fd and M13,typically M13.

In certain embodiments, an antibody or antibody fragment is maderecombinantly in a host cell. In other words, once the sequence of theantibody is known (for example, using the methods described above), theantibody can be made recombinantly using standard techniques.

In certain embodiments, the internalizing moieties may be modified tomake them more resistant to cleavage by proteases. For example, thestability of an internalizing moiety comprising a polypeptide may beincreased by substituting one or more of the naturally occurring aminoacids in the (L) configuration with D-amino acids. In variousembodiments, at least 1%, 5%, 10%, 20%, 50%, 80%, 90% or 100% of theamino acid residues of internalizing moiety may be of the Dconfiguration. The switch from L to D amino acids neutralizes thedigestion capabilities of many of the ubiquitous peptidases found in thedigestive tract. Alternatively, enhanced stability of an internalizingmoiety comprising a peptide bond may be achieved by the introduction ofmodifications of the traditional peptide linkages. For example, theintroduction of a cyclic ring within the polypeptide backbone may conferenhanced stability in order to circumvent the effect of many proteolyticenzymes known to digest polypeptides in the stomach or other digestiveorgans and in serum. In still other embodiments, enhanced stability ofan internalizing moiety may be achieved by intercalating one or moredextrorotatory amino acids (such as, dextrorotatory phenylalanine ordextrorotatory tryptophan) between the amino acids of internalizingmoiety. In exemplary embodiments, such modifications increase theprotease resistance of an internalizing moiety without affecting theactivity or specificity of the interaction with a desired targetmolecule.

(b) Homing Peptides

In certain aspects, an internalizing moiety may comprise a homingpeptide which selectively directs the subject chimeric GAA, laforin,alpha-amylase, malin and/or AGL polypeptide to a target tissue (e.g.,muscle). For example, delivering a chimeric polypeptide to the musclecan be mediated by a homing peptide comprising an amino acid sequence ofASSLNIA. Further exemplary homing peptides are disclosed in WO 98/53804.Homing peptides for a target tissue (or organ) can be identified usingvarious methods well known in the art. Additional examples of homingpeptides include the HIV transactivator of transcription (TAT) whichcomprises the nuclear localization sequence Tat48-60; Drosophilaantennapedia transcription factor homeodomain (e.g., Penetratin whichcomprises Antp43-58 homeodomain 3rd helix); Homo-arginine peptides(e.g., Arg7 peptide-PKC-c agonist protection of ischemic rat heart);alpha-helical peptides; cationic peptides (“superpositively” chargedproteins). In some embodiments, the homing peptide transits cellularmembranes via an equilibrative nucleoside (ENT) transporter. In someembodiments, the homing peptide transits cellular membranes via an ENT1,ENT2, ENT3 or ENT4 transporter. In some embodiments, the homing peptidetargets ENT2. In other embodiments, the homing peptide targets musclecells. The muscle cells targeted by the homing peptide may includeskeletal, cardiac or smooth muscle cells. In other embodiments, thehoming peptide targets neurons, epithelial cells, liver cells, kidneycells or Leydig cells.

In certain embodiments, the homing peptide is capable of bindingpolynucleotides. In certain embodiments, the homing peptide is capableof binding DNA. In certain embodiments, the homing peptide is capable ofbinding DNA with a K_(D) of less than 1 In certain embodiments, thehoming peptide is capable of binding DNA with a K_(D) of less than 100nM.

Additionally, homing peptides for a target tissue (or organ) can beidentified using various methods well known in the art. Once identified,a homing peptide that is selective for a particular target tissue can beused, in certain embodiments.

An exemplary method is the in vivo phage display method. Specifically,random peptide sequences are expressed as fusion peptides with thesurface proteins of phage, and this library of random peptides areinfused into the systemic circulation. After infusion into host mice,target tissues or organs are harvested, the phage is then isolated andexpanded, and the injection procedure repeated two more times. Eachround of injection includes, by default, a negative selection component,as the injected virus has the opportunity to either randomly bind totissues, or to specifically bind to non-target tissues. Virus sequencesthat specifically bind to non-target tissues will be quickly eliminatedby the selection process, while the number of non-specific binding phagediminishes with each round of selection. Many laboratories haveidentified the homing peptides that are selective for vasculature ofbrain, kidney, lung, skin, pancreas, intestine, uterus, adrenal gland,retina, muscle, prostate, or tumors. See, for example, Samoylova et al.,1999, Muscle Nerve, 22:460; Pasqualini et al., 1996, Nature, 380:364;Koivunen et al., 1995, Biotechnology, 13:265; Pasqualini et al., 1995,J. Cell Biol., 130:1189; Pasqualini et al., 1996, Mole. Psych., 1:421,423; Rajotte et al., 1998, J. Clin. Invest., 102:430; Rajotte et al.,1999, J. Biol. Chem., 274:11593. See, also, U.S. Pat. Nos. 5,622,699;6,068,829; 6,174,687; 6,180,084; 6,232,287; 6,296,832; 6,303,573;6,306,365. Homing peptides that target any of the above tissues may beused for targeting a GAA, laforin, alpha-amylase, malin and/or AGLprotein to that tissue.

(c) Additional Targeting to Lysosomes and Autophagic Vesicles

A traditional method of targeting a protein to lysosomes is modificationof the protein with M6P residues, which directs their transport tolysosomes through interaction of M6P residues and M6PR molecules on theinner surface of structures such as the Golgi apparatus or lateendosome. Transport of endogenous GAA, laforin, alpha-amylase, malinand/or AGL to the lysosome depends on M6P and M6PR interaction. Thereare also forms of M6P independent transport of GAA, as evidenced bynormal activity of GAA even in patients with I-cell disease, whichmanifests with severe deficiencies in other lysosomal enzymes (Wisselaret al., J. Biological Chemistry, 268(3): 2223-2231, 1993). Furtherevidence of M6P independent transport of GAA is evidenced by a studyshowing no disruption in lysosomal GAA in muscle-specific M6PR-knockoutmice targeting (Wylie et al., 2003, Am J Pathol, 162(1): 321-28). Incertain embodiments, chimeric polypeptides of the present disclosure(e.g., polypeptides comprising GAA, such as mature GAA, laforin,alpha-amylase, malin and/or AGL; and an internalizing moiety) mayfurther include modification to facilitate additional targeting to thelysosome through M6PRs or in pathways independent of M6PRs. Suchtargeting moieties may be added, for example, at the N-terminus orC-terminus of a chimeric polypeptide, and via conjugation to 3E10 orGAA, laforin, alpha-amylase, malin and/or AGL. In other embodiments, theGAA, laforin, alpha-amylase, malin and/or AGL portion of a chimericpolypeptide comprises all or some of the endogenous sequences tofacilitate M6P transport.

In some embodiments, the chimeric polypeptides of the present disclosureare transported to lysosomes via the cellular process of autophagy.Autophagy is a catabolic mechanism that involves cell degradation ofunnecessary or dysfunctional cellular components through the lysosomalmachinery. During this process, targeted cytoplasmic constituents areisolated from the rest of the cell within vesicles calledautophagosomes, which are then fused with lysosomes and degraded orrecycled. Uptake of proteins into autophagic vesicles is mediated by theformation of a membrane around the targeted region of a cell andsubsequent fusion of the vesicle with a lysosome. Several mechanisms forautophagy are known, including macroautophagy in which organelles andproteins are sequestered within the cell in a vesicle called anautophagic vacuole. Upon fusion with the lysosome, the contents of theautophagic vacuole are degraded by acidic lysosomal hydrolases. Inmicroautophagy, lysosomes engulf cytoplasm directly, and inchaperone-mediated autophagy, proteins with a consensus peptide sequenceare bound by a hsc70-containing chaperone-cochaperone complex, which isrecognized by a lysosomal protein and translocated across the lysosomalmembrane. Autophagic vacuoles have a lysosomal environment (low pH),which is conducive for activity of enzymes such as GAA (e.g., matureGAA).

Autophagy naturally occurs in muscle cells of mammals (Masiero et al,2009, Cell Metabolism, 10(6): 507-15). As the autophagic vacuoles takeup proteins from the cytoplasm, the chimeric polypeptides of the presentdisclosure are expected to be taken up by glycogen-containing autophagicvesicles, where the chimeric polypeptides would be free to degrade anyglycogen present within those vacuoles. As such, in some embodiments,the chimeric polypeptides are capable of being taken up by autophagicvacuoles without addition of any autophagic vacuole-specific targetingmotif.

In certain embodiments, the chimeric polypeptides of the presentdisclosure may further include modification to facilitate additionaltargeting to autophagic vesicles. One known chaperone-targeting motif isKFERQ-like motif. Accordingly, this motif can be added to chimericpolypeptides as described herein, in order to target the polypeptidesfor autophagy. Such targeting moieties may be added, for example, at theN-terminus or C-terminus of a chimeric polypeptide, and via conjugationto 3E10 or GAA, laforin, alpha-amylase, malin and/or AGL.

M6P residues or chaperone-targeting motifs may be added to the GAA,laforin, alpha-amylase, malin and/or AGL polypeptides.

III. Chimeric Polypeptides

The disclosure provides chimeric polypeptides comprising aninternalizing moiety portion and a non-internalizing moiety portion. Asdetailed above, the non-internalizing moiety polypeptide portioncomprises or consists of a GAA polypeptide, a laforin polypeptide,alpha-amylase polypeptide, a malin polypeptide or an AGL polypeptide.Numerous examples of internalizing moieties, and each of the potentialnon-internalizing moiety polypeptide portions are described above, andall suitable combinations of internalizing moiety portions andnon-internalizing moiety polypeptide portions to generate chimericpolypeptides are contemplated.

Without being bound by theory, regardless of whether thenon-internalizing moiety polypeptide portion of the chimeric polypeptidecomprises or consists of GAA, laforin, alpha-amylase, malin and/or AGL,its association with the internalizing moiety portion facilitatesdelivery of the chimeric polypeptide, and thus, the non-internalizingmoiety portion to the cytoplasm and, optionally, to the lysosome and/orautophagic vesicles. In certain embodiments, the internalizing moietydelivers GAA, laforin, alpha-amylase, malin and/or AGL activity intocells. In certain embodiments, the chimeric polypeptide of thedisclosure comprises a GAA-containing chimeric polypeptide (e.g., thenon-internalizing moiety portion comprises or consists of a GAApolypeptide). In certain embodiments, the chimeric polypeptide of thedisclosure comprises an AGL-containing chimeric polypeptide (e.g., thenon-internalizing moiety portion comprises or consists of an AGLpolypeptide). In certain embodiments, the chimeric polypeptide of thedisclosure comprises a laforin-containing chimeric polypeptide (e.g.,the non-internalizing moiety portion comprises or consists of a laforinpolypeptide). In certain embodiments, the chimeric polypeptide of thedisclosure comprises a malin-containing chimeric polypeptide (e.g., thenon-internalizing moiety portion comprises or consists of a malinpolypeptide). In certain embodiments, the chimeric polypeptide of thedisclosure comprises an alpha-amylase-containing chimeric polypeptide(e.g., the non-internalizing moiety portion comprises or consists of analpha-amylase polypeptide). Any of the internalizing moieties describedherein may be combined with any of the non-internalizing moietypolypeptide portions, as described herein, to generate a chimericpolypeptide of the disclosure.

The disclosure provides chimeric polypeptides (e.g., chimericpolypeptides of the disclosure). Chimeric polypeptides for use in themethods disclosed herein can be made in various manners. The chimericpolypeptides may comprise any of the internalizing moiety portions andthe GAA, laforin, alpha-amylase, malin or AGL polypeptide portionsdisclosed herein (e.g., a GAA polypeptide comprising mature GAA, asdescribed herein). As used herein, chimeric polypeptides of thedisclosure comprise (i) a GAA, laforin, alpha-amylase, malin and/or AGLpolypeptide portion and (ii) an internalizing moiety portion. Inaddition, any of the chimeric polypeptides disclosed herein may beutilized in any of the methods or compositions disclosed herein. In someembodiments, an internalizing moiety (e.g. an antibody or a homingpeptide) is linked, directly or indirectly, to any one of the GAApolypeptides (e.g., mature GAA polypeptides) , laforin, alpha-amylase,malin and/or AGL, and/or fragments or variants disclosed herein.

In some embodiments, the chimeric polypeptide comprises immature GAApolypeptide, e.g., a GAA polypeptide having the amino acid sequences ofeither SEQ ID NOs: 1 or 2. In some embodiments, the chimeric polypeptidedoes not comprise an: i) immature GAA polypeptide of approximately110kDa and/or, ii) immature GAA possessing the signal sequence, i.e.,amino acid residues 1-27 of SEQ ID NO: 1 or 2. In other words, thedisclosure contemplates chimeric polypeptides in which the chimericpolypeptide comprises a mature GAA polypeptide, but may also includeadditional polypeptide sequence from a GAA polypeptide, includingsequence contiguous with the mature GAA polypeptide (e.g., the GAApolypeptide portion comprises a GAA polypeptide comprising a mature GAApolypeptide sequence). For example, in some embodiments, the chimericpolypeptides comprise a GAA polypeptide comprising the amino acidsequence of any of SEQ ID NOs: 21-23 (e.g., SEQ ID NOs 21-23 areexemplary of GAA polypeptides comprising mature GAA but which alsoinclude additional contiguous amino acids of a GAA polypeptide). Thedisclosure also contemplates embodiments in which the chimericpolypeptide comprises a mature GAA polypeptide but does not includeadditional GAA polypeptide sequence contiguous with the mature GAApolypeptide portion. Finally, the disclosure contemplates embodiments inwhich the chimeric polypeptide does not include additional GAApolypeptide portions in addition to the mature GAA polypeptide.

In certain embodiments, it may be desirable to conjugate any of theinternalizing moieties described herein with a mature GAA polypeptide(e.g., a GAA polypeptide having the amino acid sequence of SEQ ID NO: 3or 4) in order to reduce the likelihood that a chimeric polypeptidecomprising a larger GAA polypeptide (e.g., a GAA polypeptide having theamino acid sequence of any of SEQ ID NOs: 21-23) is inadvertentlycleaved at any of the cleavage sites present in the full-length GAApolypeptide (e.g., cleaving between any of the amino acids correspondingto amino acids 56-57, 77-78, 113-114, 121-122, 200-201, 203-204,781-782, or 791-792 of SEQ ID NO: 1) by a subject's proteases prior touptake of the chimeric polypeptide by a targeted cell in the subject.

In some embodiments, the chimeric polypeptides comprise an amino acidsequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity to anyof SEQ ID NOs: 38-45, or biologic fragments thereof.

In certain embodiments, the C-terminus of a GAA polypeptide (e.g., amature GAA polypeptide), a laforin polypeptide, alpha-amylasepolypeptide, malin polypeptide and/or an AGL polypeptide can be linked,directly or indirectly, to the N-terminus of an internalizing moiety(e.g., an antibody, an antibody fragment, or a homing peptide).Alternatively, the C-terminus of an internalizing moiety (e.g., anantibody, an antibody fragment, or a homing peptide) can be linked,directly or indirectly, to the N-terminus of a GAA, laforin,alpha-amylase, malin and/or AGL polypeptide. For example, chimericpolypeptides can be designed to place the GAA, laforin, alpha-amylase,malin and/or AGL polypeptide at the amino or carboxy terminus of eitherthe antibody heavy or light chain of mAb 3E10. In some embodiments, theGAA polypeptide comprises the amino acid sequence of SEQ ID NO: 22 or 23fused to the C-terminus of an internalizing moiety. In some embodiments,the GAA polypeptide comprises the amino acid sequence of SEQ ID NO: 22or 23 fused to the C-terminus of the heavy chain segment of a Fabinternalizing moiety. In some embodiments, the GAA polypeptide comprisesthe amino acid sequence of SEQ ID NO: 22 or 23 fused to the C-terminusof the heavy chain segment of a full-length antibody internalizingmoiety.

In some embodiments, the laforin polypeptide comprises the amino acidsequence of SEQ ID NO: 38 or 39, or variants or fragments thereof, fusedto the C-terminus of an internalizing moiety. In some embodiments, thelaforin polypeptide comprises the amino acid sequence of SEQ ID NO: 38or 39, or variants or fragments thereof, fused to the C-terminus of theheavy chain segment of a Fab internalizing moiety. In some embodiments,the laforin polypeptide comprises the amino acid sequence of SEQ ID NO:38 or 39, or variants or fragments thereof, fused to the C-terminus ofthe heavy chain segment of a full-length antibody internalizing moiety.

In some embodiments, the AGL polypeptide comprises the amino acidsequence of any of SEQ ID NO: 40-42, or variants or fragments thereof,fused to the C-terminus of an internalizing moiety. In some embodiments,the AGL polypeptide comprises the amino acid sequence of any of SEQ IDNOs: 40-42, or variants or fragments thereof, fused to the C-terminus ofthe heavy chain segment of a Fab internalizing moiety. In someembodiments, the AGL polypeptide comprises the amino acid sequence ofany of SEQ ID NOs: 40-42, or variants or fragments thereof, fused to theC-terminus of the heavy chain segment of a full-length antibodyinternalizing moiety.

In some embodiments, the malin polypeptide comprises the amino acidsequence of SEQ ID NO: 43, or variants or fragments thereof, fused tothe C-terminus of an internalizing moiety. In some embodiments, themalin polypeptide comprises the amino acid sequence of SEQ ID NO: 43, orvariants or fragments thereof, fused to the C-terminus of the heavychain segment of a Fab internalizing moiety. In some embodiments, themalin polypeptide comprises the amino acid sequence of SEQ ID NO: 43, orvariants or fragments thereof, fused to the C-terminus of the heavychain segment of a full-length antibody internalizing moiety.

In some embodiments, the alpha-amylase polypeptide comprises the aminoacid sequence of SEQ ID NO: 44 or 45, or variants or fragments thereof,fused to the C-terminus of an internalizing moiety. In some embodiments,the alpha-amylase polypeptide comprises the amino acid sequence of SEQID NO: 44 or 45, or variants or fragments thereof, fused to theC-terminus of the heavy chain segment of a Fab internalizing moiety. Insome embodiments, the alpha-amylase polypeptide comprises the amino acidsequence of SEQ ID NO: 44 or 45, or variants or fragments thereof, fusedto the C-terminus of the heavy chain segment of a full-length antibodyinternalizing moiety.

In certain embodiments, potential configurations include the use oftruncated portions of an antibody's heavy and light chain sequences(e.g., mAB 3E10) as needed to maintain the functional integrity of theattached mature GAA polypeptide. Further still, the internalizing moietycan be linked to an exposed internal (non-terminus) residue of GAA(e.g., mature GAA), laforin, alpha-amylase, malin and/or AGL or avariant thereof. In further embodiments, any combination of theGAA-internalizing moiety configurations can be employed, therebyresulting in a GAA: internalizing moiety ratio that is greater than 1:1(e.g., two mature GAA molecules to one internalizing moiety). In furtherembodiments, any combination of the laforin-internalizing moietyconfigurations can be employed, thereby resulting in alaforin:internalizing moiety ratio that is greater than 1:1 (e.g., twolaforin molecules to one internalizing moiety). In further embodiments,any combination of the AGL-internalizing moiety configurations can beemployed, thereby resulting in a AGL:internalizing moiety ratio that isgreater than 1:1 (e.g., two AGL molecules to one internalizing moiety).In further embodiments, any combination of the malin-internalizingmoiety configurations can be employed, thereby resulting in amalin:internalizing moiety ratio that is greater than 1:1 (e.g., twomalin molecules to one internalizing moiety). In further embodiments,any combination of the alpha-amylase-internalizing moiety configurationscan be employed, thereby resulting in a alpha-amylase:internalizingmoiety ratio that is greater than 1:1 (e.g., two alpha-amylase moleculesto one internalizing moiety).

The GAA polypeptide (e.g., mature GAA polypeptide), laforin polypeptide,alpha-amylase polypeptide, malin polypeptide and/or AGL polypeptide andthe internalizing moiety may be linked directly to each other.Alternatively, they may be linked to each other via a linker sequence,which separates the GAA polypeptide, laforin polypeptide, alpha-amylasepolypeptide, malin polypeptide and/or AGL polypeptide and theinternalizing moiety by a distance sufficient to ensure that each domainproperly folds into its secondary and tertiary structures. Preferredlinker sequences (1) should adopt a flexible extended conformation, (2)should not exhibit a propensity for developing an ordered secondarystructure which could interact with the functional domains of the GAApolypeptide, laforin polypeptide, alpha-amylase polypeptide, malinpolypeptide and/or AGL polypeptide or the internalizing moiety, and (3)should have minimal hydrophobic or charged character, which couldpromote interaction with the functional protein domains. Typical surfaceamino acids in flexible protein regions include Gly, Asn and Ser.Permutations of amino acid sequences containing Gly, Asn and Ser wouldbe expected to satisfy the above criteria for a linker sequence. Othernear neutral amino acids, such as Thr and Ala, can also be used in thelinker sequence. In a specific embodiment, a linker sequence length ofabout 20 amino acids can be used to provide a suitable separation offunctional protein domains, although longer or shorter linker sequencesmay also be used. The length of the linker sequence separating the GAA,laforin, alpha-amylase, AGL and/or malin polypeptide from theinternalizing moiety can be from 5 to 500 amino acids in length, or morepreferably from 5 to 100 amino acids in length. Preferably, the linkersequence is from about 5-30 amino acids in length. In preferredembodiments, the linker sequence is from about 5 to about 20 aminoacids, and is advantageously from about 10 to about 20 amino acids. Inother embodiments, the linker joining the GAA polypeptide, laforinpolypeptide, alpha-amylase polypeptide, malin polypeptide and/or AGLpolypeptide to an internalizing moiety can be a constant domain of anantibody (e.g., constant domain of mAb 3E10 or all or a portion of an Fcregion of another antibody). In certain embodiments, the linker is acleavable linker. In certain embodiments, the linker sequence comprisesthe linker sequence of SEQ ID NO: 30. In certain embodiments, theinternalizing moiety is an antibody or antibody fragment and theconjugation includes chemical or recombinant conjugation to a constantdomain, such as the constant domain of a heavy chain of the antibody orantibody fragment. In such embodiments, it is appreciated that the GAA,laforin, alpha-amylase, AGL and/or malin polypeptide and internalizingmoiety may be further associated via the association between the heavychain and light chain of the antibody or antibody fragment. This is alsoincluded within the scope of the conjugation.

In other embodiments, the GAA polypeptide, laforin polypeptide,alpha-amylase polypeptide, malin polypeptide and/or AGL polypeptide orfunctional fragment thereof may be conjugated or joined directly to theinternalizing moiety. For example, a recombinantly conjugated chimericpolypeptide can be produced as an in-frame fusion of the GAA, laforin,alpha-amylase, malin and/or AGL portion and the internalizing moietyportion. In certain embodiments, the linker may be a cleavable linker.In any of the foregoing embodiments, the internalizing moiety may beconjugated (directly or via a linker) to the N-terminal or C-terminalamino acid of the GAA polypeptide, laforin polypeptide, alpha-amylasepolypeptide, malin polypeptide and/or AGL polypeptide, such as to theN-terminal or C-terminal amino acid of a GAA polypeptide comprising amature GAA. In other embodiments, the internalizing moiety may beconjugated (directly or indirectly) to an internal amino acid of the GAApolypeptide, laforin polypeptide, alpha-amylase polypeptide, malinpolypeptide and/or AGL polypeptide. Note that the two portions of theconstruct are conjugated/joined to each other. Unless otherwisespecified, describing the chimeric polypeptide as a conjugation of theGAA portion to the internalizing moiety is used equivalently as aconjugation of the internalizing moiety to the GAA portion. Unlessotherwise specified, describing the chimeric polypeptide as aconjugation of the laforin portion to the internalizing moiety is usedequivalently as a conjugation of the internalizing moiety to the laforinportion. Unless otherwise specified, describing the chimeric polypeptideas a conjugation of the AGL portion to the internalizing moiety is usedequivalently as a conjugation of the internalizing moiety to the AGLportion. Unless otherwise specified, describing the chimeric polypeptideas a conjugation of the malin portion to the internalizing moiety isused equivalently as a conjugation of the internalizing moiety to themalin portion. Unless otherwise specified, describing the chimericpolypeptide as a conjugation of the alpha-amylase portion to theinternalizing moiety is used equivalently as a conjugation of theinternalizing moiety to the alpha-amylase portion. Further, unlessotherwise specified, conjugation and/or joining refers to eitherchemical or genetic conjugation.

In certain embodiments, the chimeric polypeptides of the presentdisclosure can be generated using well-known cross-linking reagents andprotocols. For example, there are a large number of chemicalcross-linking agents that are known to those skilled in the art anduseful for cross-linking the GAA polypeptide, laforin polypeptide,alpha-amylase polypeptide, malin polypeptide and/or AGL polypeptide withan internalizing moiety (e.g., an antibody). For example, thecross-linking agents are heterobifunctional cross-linkers, which can beused to link molecules in a stepwise manner. Heterobifunctionalcross-linkers provide the ability to design more specific couplingmethods for conjugating proteins, thereby reducing the occurrences ofunwanted side reactions such as homo-protein polymers. A wide variety ofheterobifunctional cross-linkers are known in the art, includingsuccinimidyl 4-(N-maleimidomethyl) cyclohexane- 1-carboxylate (SMCC),m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC);4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-tolune (SMPT),N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl6-[3-(2-pyridyldithio) propionate] hexanoate (LC-SPDP). Thosecross-linking agents having N-hydroxysuccinimide moieties can beobtained as the N-hydroxysulfosuccinimide analogs, which generally havegreater water solubility. In addition, those cross-linking agents havingdisulfide bridges within the linking chain can be synthesized instead asthe alkyl derivatives so as to reduce the amount of linker cleavage invivo. In addition to the heterobifunctional cross-linkers, there existsa number of other cross-linking agents including homobifunctional andphotoreactive cross-linkers. Disuccinimidyl subcrate (DSS),bismaleimidohexane (BMH) and dimethylpimelimidate.2 HCl (DMP) areexamples of useful homobifunctional cross-linking agents, and bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) andN-succinimidyl-6(4′-azido-2′-nitrophenylamino)hexanoate (SANPAH) areexamples of useful photoreactive cross-linkers for use in thisdisclosure. For a recent review of protein coupling techniques, seeMeans et al. (1990) Bioconjugate Chemistry. 1:2-12, incorporated byreference herein.

One particularly useful class of heterobifunctional cross-linkers,included above, contain the primary amine reactive group,N-hydroxysuccinimide (NHS), or its water soluble analogN-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysine epsilongroups) at alkaline pH's are unprotonated and react by nucleophilicattack on NHS or sulfo-NHS esters. This reaction results in theformation of an amide bond, and release of NHS or sulfo-NHS as aby-product. Another reactive group useful as part of aheterobifunctional cross-linker is a thiol reactive group. Common thiolreactive groups include maleimides, halogens, and pyridyl disulfides.Maleimides react specifically with free sulfhydryls (cysteine residues)in minutes, under slightly acidic to neutral (pH 6.5-7.5) conditions.Halogens (iodoacetyl functions) react with --SH groups at physiologicalpH's. Both of these reactive groups result in the formation of stablethioether bonds. The third component of the heterobifunctionalcross-linker is the spacer arm or bridge. The bridge is the structurethat connects the two reactive ends. The most apparent attribute of thebridge is its effect on steric hindrance. In some instances, a longerbridge can more easily span the distance necessary to link two complexbiomolecules.

In some embodiments, the chimeric polypeptide comprises multiplelinkers. For example, if the chimeric polypeptide comprises an scFvinternalizing moiety, the chimeric polypeptide may comprise a firstlinker conjugating the GAA, laforin, alpha-amylase, AGL and/or malin tothe internalizing moiety, and a second linker in the scFv conjugatingthe V_(H) domain (e.g., SEQ ID NO: 9) to the V_(L) domain (e.g., SEQ IDNO: 10).

Preparing protein-conjugates using heterobifunctional reagents is atwo-step process involving the amine reaction and the sulfhydrylreaction. For the first step, the amine reaction, the protein chosenshould contain a primary amine. This can be lysine epsilon amines or aprimary alpha amine found at the N-terminus of most proteins. Theprotein should not contain free sulfhydryl groups. In cases where bothproteins to be conjugated contain free sulfhydryl groups, one proteincan be modified so that all sulfhydryls are blocked using for instance,N-ethylmaleimide (see Partis et al. (1983) J. Pro. Chem. 2:263,incorporated by reference herein). Ellman's Reagent can be used tocalculate the quantity of sulfhydryls in a particular protein (see forexample Ellman et al. (1958) Arch. Biochem. Biophys. 74:443 and Riddleset al. (1979) Anal. Biochem. 94:75, incorporated by reference herein).

In certain specific embodiments, chimeric polypeptides of the disclosurecan be produced by using a universal carrier system. For example, a GAApolypeptide, laforin polypeptide, alpha-amylase polypeptide, malinpolypeptide and/or AGL polypeptide can be conjugated to a common carriersuch as protein A, poly-L-lysine, hex-histidine, and the like. Theconjugated carrier will then form a complex with an antibody which actsas an internalizing moiety. A small portion of the carrier molecule thatis responsible for binding immunoglobulin could be used as the carrier.

In certain embodiments, chimeric polypeptides of the disclosure can beproduced by using standard protein chemistry techniques such as thosedescribed in Bodansky, M. Principles of Peptide Synthesis, SpringerVerlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: AUser's Guide, W. H. Freeman and Company, New York (1992). In addition,automated peptide synthesizers are commercially available (e.g.,Advanced ChemTech Model 396; Milligen/Biosearch 9600). In any of theforegoing methods of cross-linking for chemical conjugation of laforin,alpha-amylase, malin, AGL and/or mature GAA to an internalizing moiety,a cleavable domain or cleavable linker can be used. Cleavage will allowseparation of the internalizing moiety and the GAA, laforin,alpha-amylase, AGL and/or malin polypeptide. For example, followingpenetration of a cell by a chimeric polypeptide, cleavage of thecleavable linker would allow separation of GAA, laforin, alpha-amylase,malin and/or AGL from the internalizing moiety.

In certain embodiments, the chimeric polypeptides comprising a GAApolypeptide portion (e.g., a GAA polypeptide comprising a mature GAApolypeptide sequence), laforin polypeptide, alpha-amylase polypeptide,malin polypeptide and/or AGL polypeptide and an internalizing moietyportion can be generated as a fusion protein containing the GAApolypeptide, laforin polypeptide, alpha-amylase polypeptide, malinpolypeptide and/or AGL polypeptide and the internalizing moiety. Incertain embodiments, the chimeric polypeptides of the present disclosurecan be generated as a fusion protein containing a GAA, laforin,alpha-amylase, AGL and/or malin polypeptide and an internalizing moiety(e.g., an antibody or a homing peptide), expressed as one contiguouspolypeptide chain. In certain embodiments, the chimeric polypeptide isgenerated as a fusion protein that comprises a GAA polypeptide portionand internalizing moiety portion, wherein the GAA polypeptide portioncomprises a mature GAA polypeptide and also includes additionalpolypeptide sequence from a GAA polypeptide, including sequencecontiguous with the mature GAA polypeptide. In certain embodiments, thechimeric polypeptide is generated as a fusion protein that comprises alaforin polypeptide portion and internalizing moiety portion. In certainembodiments, the chimeric polypeptide is generated as a fusion proteinthat comprises an AGL polypeptide portion and internalizing moietyportion. In certain embodiments, the chimeric polypeptide is generatedas a fusion protein that comprises a malin polypeptide portion andinternalizing moiety portion. In certain embodiments, the chimericpolypeptide is generated as a fusion protein that comprises analpha-amylase polypeptide portion and internalizing moiety portion. Inpreparing such fusion protein, a fusion gene is constructed comprisingnucleic acids which encode a laforin polypeptide, alpha-amylasepolypeptide, malin polypeptide an AGL polypeptide and/or a mature GAApolypeptide, and an internalizing moiety, and optionally, a peptidelinker sequence to span the GAA, laforin, alpha-amylase, AGL and/ormalin polypeptide and the internalizing moiety. The use of recombinantDNA techniques to create a fusion gene, with the translational productbeing the desired fusion protein, is well known in the art. Both thecoding sequence of a gene and its regulatory regions can be redesignedto change the functional properties of the protein product, the amountof protein made, or the cell type in which the protein is produced. Thecoding sequence of a gene can be extensively altered—for example, byfusing part of it to the coding sequence of a different gene to producea novel hybrid gene that encodes a fusion protein. Examples of methodsfor producing fusion proteins are described in PCT applicationsPCT/US87/02968, PCT/US89/03587 and PCT/US90/07335, as well as Trauneckeret al. (1989) Nature 339:68, incorporated by reference herein.Essentially, the joining of various DNA fragments coding for differentpolypeptide sequences is performed in accordance with conventionaltechniques, employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini, fillingin of cohesive ends as appropriate, alkaline phosphatase treatment toavoid undesirable joining, and enzymatic ligation. Alternatively, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. In another method, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, Eds. Ausubel et al.John Wiley & Sons: 1992). The chimeric polypeptides encoded by thefusion gene may be recombinantly produced using various expressionsystems as is well known in the art (also see below).

Recombinantly conjugated chimeric polypeptides include embodiments inwhich the GAA polypeptide, laforin polypeptide, alpha-amylasepolypeptide, malin polypeptide and/or AGL polypeptide is conjugated tothe N-terminus or C-terminus of the internalizing moiety. Exemplarychimeric polypeptides in which GAA, laforin, alpha-amylase, AGL and/ormalin polypeptides are conjugated to variant light and heavy chains ofFv3E10 are indicated in SEQ ID NOs: 11 and 12. In certain embodiments, achimeric polypeptide of the disclosure further comprises, at theN-terminus (at or within 10 amino acid residues of the N-terminus), anamino acid sequence set forth in SEQ ID NO: 19 or 20.

Recombinantly conjugated chimeric polypeptides include embodiments inwhich the internalizing moiety is N-terminal to the GAA polypeptide,laforin polypeptide, alpha-amylase polypeptide, malin polypeptide and/orAGL polypeptide and embodiments in which the internalizing moiety isC-terminal to the GAA, laforin, alpha-amylase, AGL and/or malinpolypeptide portion. We note that methods of making fusion proteinsrecombinantly are well known in the art. Any of the chimeric proteinsdescribed herein can readily be made recombinantly. This includesproteins having one or more tags and/or one or more linkers. Forexample, if the chimeric polypeptide comprises an scFv internalizingmoiety, the chimeric polypeptide may comprise a first linkerinterconnection the internalizing moiety to the GAA polypeptide, laforinpolypeptide, alpha-amylase polypeptide, malin polypeptide and/or AGLpolypeptide portion, and a second linker in the scFv conjugating theV_(H) domain. Moreover, in certain embodiments, the chimericpolypeptides comprise a “AGIH” portion (SEQ ID NO: 19) on the N-terminusof the chimeric polypeptide (or within 10 amino acid residues of theN-terminus), and such chimeric polypeptides may be provided in thepresence or absence of one or more epitope tags. In further embodiments,the chimeric polypeptide comprises a serine at the N-terminal mostposition of the polypeptide. In some embodiments, the chimericpolypeptides comprise an “SAGIH” (SEQ ID NO: 20) portion at theN-terminus of the polypeptide (or within 10 amino acid residues of theN-terminus), and such chimeric polypeptides may be provided in thepresence or absence of one or more epitope tags.

In some embodiments, the chimeric polypeptides comprise a signalsequence (e.g., SEQ ID NO: 33 or 36). In some embodiments, the signalsequence (e.g., SEQ ID NO: 33) is at the N-terminus of the light chainsequence of any of the antibodies or antigen binding fragments disclosedherein. In some embodiments, the signal sequence (e.g., SEQ ID NO: 33)is at the N-terminus of the amino acid sequence SEQ ID NO: 10, orfragments or variants thereof. In some embodiments, the signal sequence(e.g., SEQ ID NO: 36) is at the N-terminus of the heavy chain sequenceof any of the antibodies or antigen binding fragments disclosed herein.In some embodiments, the signal sequence (e.g., SEQ ID NO: 36) is at theN-terminus of the amino acid sequence SEQ ID NO: 9, or fragments orvariants thereof.

In some embodiments, the chimeric polypeptides are producedrecombinantly in cells. In some embodiments, the cells are bacteria(e.g., E. coli), yeast (e.g., Picchia), insect cells (e.g., Sf9 cells)or mammalian cells (e.g., CHO or HEK-293 cells). Chimeric polypeptidesof the disclosure are, in certain embodiments, made in any of theforegoing cells in culture using art recognized techniques for makingand purifying protein from cells or cell supernatant.

The presence in the chimeric polypeptide of all or a portion of animmunoglobulin or an epitope tag, such as an HA or myc tag, provides aregion for purification of chimeric polypeptide. In some embodiments, atag or the immunoglobulin portion of the chimeric polypeptide are usedfor purification such that a composition comprising a chimericpolypeptide of the disclosure is enriched and or substantially purifiedrelative to GAA portions that are not interconnected to an internalizingmoiety portion. For example, the presence of GAA is enriched such thatgreater than 90% of the GAA in a composition is presented as apolypeptide interconnected to an internalizing moiety. In otherembodiments, the composition is enriched such that greater than 80%,greater than 85%, greater than 90% or greater than 95% of the GAA in acomposition is approximately the same molecular weight and/or differs atthe N-terminus of the GAA portion by less than 5 amino acid residues.

In some embodiments, the immunogenicity of the chimeric polypeptide maybe reduced by identifying a candidate T-cell epitope within a junctionregion spanning the chimeric polypeptide and changing an amino acidwithin the junction region as described in U.S. Patent Publication No.2003/0166877.

Chimeric polypeptides according to the disclosure can be used fornumerous purposes. We note that any of the chimeric polypeptidesdescribed herein can be used in any of the methods described herein, andsuch suitable combinations are specifically contemplated.

Chimeric polypeptides described herein can be used to deliver GAA,laforin, alpha-amylase, malin and/or AGL polypeptide to cells,particular to a muscle cell. In certain embodiments, chimericpolypeptides deliver GAA (e.g., mature GAA), laforin, alpha-amylase,malin and/or AGL to liver cells. Thus, the chimeric polypeptides can beused to facilitate transport of GAA, laforin, alpha-amylase, malin,and/or AGL to cells in vitro or in vivo. By facilitating transport tocells, the chimeric polypeptides improve delivery efficiency, thusfacilitating working with GAA, laforin, alpha-amylase, malin and/or AGLpolypeptide in vitro or in vivo. Further, by increasing the efficiencyof transport, the chimeric polypeptides may help decrease the amount ofGAA, laforin, alpha-amylase, malin and/or AGL needed for in vitro or invivo experimentation. Moreover, by facilitating delivery to thecytoplasm, the chimeric polypeptides and methods of the disclosure canaddress the problems associated with cytoplasmic accumulation ofglycogen in, for example, Forbes-Cori and/or Andersen Disease and/orPompe Disease and/or von Gierke Disease and/or Lafora Disease.

The chimeric polypeptides can be used to study the function of GAA(e.g., mature GAA), laforin, alpha-amylase, malin and/or AGL in cells inculture, as well as to study transport of GAA, laforin, alpha-amylase,malin and/or AGL. The chimeric polypeptides can be used to identifybinding partners for GAA, laforin, alpha-amylase, malin and/or AGL incells, such as transport between cytoplasm and lysosome. The chimericpolypeptides can be used in screens to identify modifiers (e.g., smallorganic molecules or polypeptide modifiers) of GAA, laforin,alpha-amylase, malin and/or AGL activity in a cell. The chimericpolypeptides can be used to help treat or alleviate the symptoms ofForbes-Cori and/or Andersen Disease (and/or Pompe Disease and/or vonGierke Disease and/or Lafora Disease) in humans or in an animal model.The foregoing are merely exemplary of the uses for the subject chimericpolypeptides.

Any of the chimeric polypeptides described herein, including chimericpolypeptides combining any of the features of the GAA polypeptides,internalizing moieties, and linkers, may be used in any of the methodsof the disclosure.

IV. GAA-Related Nucleic Acids and Expression

In certain embodiments, the present disclosure makes use of nucleicacids for producing a GAA polypeptide, e.g., mature GAA polypeptide(including functional fragments, variants, and fusions thereof), such asfor producing GAA polypeptides comprising a mature GAA polypeptide. Incertain embodiments, the present disclosure makes use of nucleic acidsfor producing a laforin polypeptide (including functional fragments,variants, and fusions thereof). In certain embodiments, the presentdisclosure makes use of nucleic acids for producing an AGL polypeptide(including functional fragments, variants, and fusions thereof). Incertain embodiments, the present disclosure makes use of nucleic acidsfor producing a malin polypeptide (including functional fragments,variants, and fusions thereof). In certain embodiments, the presentdisclosure makes use of nucleic acids for producing an alpha-amylasepolypeptide (including functional fragments, variants, and fusionsthereof). In certain specific embodiments, the nucleic acids may furthercomprise DNA which encodes an internalizing moiety (e.g., an antibody ora homing peptide) for making a recombinant chimeric protein of thedisclosure.

In certain embodiments, the nucleic acid construct does not encode achimeric polypeptide comprising a GAA precursor polypeptide ofapproximately 110 kDa. In certain embodiments, the nucleic acidconstruct encodes a GAA polypeptide comprising immature GAA polypeptide(e.g., a GAA polypeptide having the amino acid sequence of SEQ ID NOs: 1or 2). In other embodiments, the nucleic acid construct encodes a GAApolypeptide comprising mature GAA but does not encode a GAA polypeptidecomprising (i) the amino acid sequence set forth in SEQ ID NO: 1 or 2 or(ii) a portion corresponding to residues 1-27 and/or 1-56 of SEQ ID NO:1 or 2. All these nucleic acids are collectively referred to as matureGAA nucleic acids because they encode a polypeptide comprising a matureGAA polypeptide and, optionally, additional contiguous portions of a GAApolypeptide.

The nucleic acids may be single-stranded or double-stranded, DNA or RNAmolecules. In certain embodiments, the disclosure relates to isolated orrecombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to a region of a GAA nucleotide sequence(e.g., GenBank Accession No.: NM_000152.3 which encodes NP000143.2;NM_001079803.1 which encodes NP_001073271.1; and NM_001079804.1 whichencodes NP_001073272.1). In certain embodiments, the GAA nucleotideencodes mature GAA (e.g., mature GAA nucleotide sequence). In certainembodiments, the disclosure relates to isolated or recombinant nucleicacid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to a region of a laforin nucleotide sequence (e.g.,GenBank Accession No. NM_005670.3 or GenBank Accession No.NM_001018041.1). In certain embodiments, the disclosure relates toisolated or recombinant nucleic acid sequences that are at least 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to a region of an AGLnucleotide sequence (e.g., GenBank Accession Number-NM_000642; GenBankAccession Number-NM_000644; GenBank Accession Number-NM_000643; GenBankAccession Number-NM_000028; GenBank Accession Number-NM_000645; orGenBank Accession Number-NM_000646). In certain embodiments, thedisclosure relates to isolated or recombinant nucleic acid sequencesthat are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical toa region of a malin nucleotide sequence (e.g., GenBank Accession No.AY324850.1). In certain embodiments, the disclosure relates to isolatedor recombinant nucleic acid sequences that are at least 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to a region of an alpha-amylasenucleotide sequence (e.g., GenBank Accession No. AH002672.1 orAH002671.1). In further embodiments, the GAA, laforin, alpha-amylase,AGL and/or malin nucleic acid sequences can be isolated, recombinant,and/or fused with a heterologous nucleotide sequence, or in a DNAlibrary.

In certain embodiments, GAA (e.g., mature GAA) , laforin, alpha-amylase,AGL and/or malin nucleic acids also include nucleotide sequences thathybridize under highly stringent conditions to any of theabove-mentioned nucleotide sequences, or complement sequences thereof.One of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. For example, one could perform the hybridization at 6.0 x sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50 ° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50 ° C. to ahigh stringency of about 0.2×SSC at 50 ° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22 ° C., to high stringency conditions at about 65 °C. Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the disclosure provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the native GAA (e.g., matureGAA) , laforin, alpha-amylase, AGL and/or malin nucleic acids due todegeneracy in the genetic code are also within the scope of thedisclosure. For example, a number of amino acids are designated by morethan one triplet. Codons that specify the same amino acid, or synonyms(for example, CAU and CAC are synonyms for histidine) may result in“silent” mutations which do not affect the amino acid sequence of theprotein. However, it is expected that DNA sequence polymorphisms that dolead to changes in the amino acid sequences of the subject proteins willexist among mammalian cells. One skilled in the art will appreciate thatthese variations in one or more nucleotides (up to about 3-5% of thenucleotides) of the nucleic acids encoding a particular protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this disclosure.

In certain embodiments, the recombinant GAA (e.g., mature GAA), laforin,alpha-amylase, AGL and/or malin nucleic acids may be operably linked toone or more regulatory nucleotide sequences in an expression construct.Regulatory nucleotide sequences will generally be appropriate for a hostcell used for expression. Numerous types of appropriate expressionvectors and suitable regulatory sequences are known in the art for avariety of host cells. Typically, said one or more regulatory nucleotidesequences may include, but are not limited to, promoter sequences,leader or signal sequences, ribosomal binding sites, transcriptionalstart and termination sequences, translational start and terminationsequences, and enhancer or activator sequences. Constitutive orinducible promoters as known in the art are contemplated by thedisclosure. The promoters may be either naturally occurring promoters,or hybrid promoters that combine elements of more than one promoter. Anexpression construct may be present in a cell on an episome, such as aplasmid, or the expression construct may be inserted in a chromosome. Ina preferred embodiment, the expression vector contains a selectablemarker gene to allow the selection of transformed host cells. Selectablemarker genes are well known in the art and will vary with the host cellused. In certain aspects, this disclosure relates to an expressionvector comprising a nucleotide sequence encoding a GAA polypeptide(e.g., mature GAA polypeptide), laforin polypeptide, alpha-amylasepolypeptide, AGL polypeptide and/or malin polypeptide, such as any ofthe GAA polypeptides, laforin polypeptides, alpha-amylase polypeptides,AGL polypeptides, and/or malin polypeptides described herein, andoperably linked to at least one regulatory sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe encoded polypeptide. Accordingly, the term regulatory sequenceincludes promoters, enhancers, and other expression control elements.Exemplary regulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology, Academic Press, San Diego, Calif.(1990). It should be understood that the design of the expression vectormay depend on such factors as the choice of the host cell (e.g., ChineseHamster Ovary cells) to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

In some embodiments, a nucleic acid construct, comprising a nucleotidesequence that encodes a GAA polypeptide (e.g., mature GAA polypeptide),laforin polypeptide, alpha-amylase polypeptide, AGL polypeptide and/ormalin polypeptide or a bioactive fragment thereof, is operably linked toa nucleotide sequence that encodes an internalizing moiety, wherein thenucleic acid construct encodes a chimeric polypeptide having GAA,laforin, alpha-amylase, AGL and/or malin biological activity. In certainembodiments, the nucleic acid constructs may further comprise anucleotide sequence that encodes a linker.

This disclosure also pertains to a host cell transfected with arecombinant gene which encodes a GAA polypeptide (e.g., mature GAApolypeptide), laforin polypeptide, alpha-amylase polypeptide, AGLpolypeptide and/or malin polypeptide or a chimeric polypeptide of thedisclosure. The host cell may be any prokaryotic or eukaryotic cell. Forexample, a GAA, laforin, alpha-amylase, AGL and/or malin polypeptide ora chimeric polypeptide may be expressed in bacterial cells such as E.coli, insect cells (e.g., using a baculovirus expression system), yeast,or mammalian cells. Other suitable host cells are known to those skilledin the art.

The present disclosure further pertains to methods of producing a GAApolypeptide (e.g., mature GAA polypeptide) , laforin polypeptide,alpha-amylase polypeptide, AGL polypeptide and/or malin polypeptide or achimeric polypeptide of the disclosure. For example, a host celltransfected with an expression vector encoding a GAA, laforin,alpha-amylase, AGL and/or malin polypeptide or a chimeric polypeptidecan be cultured under appropriate conditions to allow expression of thepolypeptide to occur. The polypeptide may be secreted and isolated froma mixture of cells and medium containing the polypeptides.Alternatively, the polypeptides may be retained cytoplasmically or in amembrane fraction and the cells harvested, lysed and the proteinisolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The polypeptides can be isolated from cell culture medium, host cells,or both using techniques known in the art for purifying proteins,including ion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for particular epitopes of the polypeptides (e.g., aGAA, laforin, alpha-amylase, AGL and/or malin polypeptide). In apreferred embodiment, the polypeptide is a fusion protein containing adomain which facilitates its purification.

A recombinant GAA (e.g., mature GAA), laforin, alpha-amylase, AGL and/ormalin nucleic acid can be produced by ligating the cloned gene, or aportion thereof, into a vector suitable for expression in eitherprokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian),or both. Expression vehicles for production of a recombinant polypeptideinclude plasmids and other vectors. For instance, suitable vectorsinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli. Thepreferred mammalian expression vectors contain both prokaryoticsequences to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papilloma virus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press, 1989) Chapters 16 and 17. In someinstances, it may be desirable to express the recombinant polypeptide bythe use of a baculovirus expression system. Examples of such baculovirusexpression systems include pVL-derived vectors (such as pVL1392, pVL1393and pVL941), pAcUW-derived vectors (such as pAcUW1), andpBlueBac-derived vectors (such as the ß-gal containing pBlueBac III).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

The disclosure contemplates methods of producing chimeric proteinsrecombinantly, such as described above. Suitable vectors and host cellsmay be readily selected for expression of proteins in, for example,yeast or mammalian cells. Host cells may express a vector encoding achimeric polypeptide stably or transiently. Such host cells may becultured under suitable conditions to express chimeric polypeptide whichcan be readily isolated from the cell culture medium.

Chimeric polypeptides of the disclosure (e.g., polypeptides comprising aGAA portion comprising mature GAA and an internalizing moiety portion)may be expressed as a single polypeptide chain or as more than onepolypeptide chains. An example of a single polypeptide chain is when aGAA, laforin, alpha-amylase, AGL and/or malin portion is fused inframeto an internalizing moiety, which internalizing moiety is an scFv. Incertain embodiments, this single polypeptide chain is expressed from asingle vectors as a fusion protein.

An example of more than one polypeptide chains is when the internalizingmoiety is an antibody or Fab. In certain embodiments, the heavy andlight chains of the antibody or Fab may be expressed in a host cellexpressing a single vector or two vectors (one expressing the heavychain and one expressing the light chain). In either case, the GAApolypeptide (e.g., mature GAA polypeptide) , laforin polypeptide,alpha-amylase polypeptide, AGL polypeptide and/or malin polypeptide maybe expressed as an inframe fusion to, for example, the C-terminus of theheavy chain such that the GAA, laforin, alpha-amylase, AGL and/or malinpolypeptide is appended to the internalizing moiety but at a distance tothe antigen binding region of the internalizing moiety.

As noted above, methods for recombinantly expressing polypeptides,including chimeric polypeptides, are well known in the art. Nucleotidesequences expressing a GAA, laforin, alpha-amylase, AGL and/or malinpolypeptide, such as a human GAA, laforin, alpha-amylase, AGL and/ormalin polypeptide, having a particular amino acid sequence are availableand can be used. Moreover, nucleotide sequences expressing aninternalizing moiety portion, such as expressing a 3E10 antibody, scFv,or Fab comprising the VH and VL set forth in SEQ ID NO: 9 and 10) arepublicly available and can be combined with nucleotide sequence encodingsuitable heavy and light chain constant regions. The disclosurecontemplates nucleotide sequences encoding any of the chimericpolypeptides of the disclosure, vectors (single vector or set ofvectors) comprising such nucleotide sequences, host cells comprisingsuch vectors, and methods of culturing such host cells to expresschimeric polypeptides of the disclosure.

V. Methods of Treatment and Other Methods of Use

For any of the methods described herein, the disclosure contemplates theuse of any of the chimeric polypeptides and/or compositions describedthroughout the application. In addition, for any of the methodsdescribed herein, the disclosure contemplates the combination of anystep or steps of one method with any step or steps from another method.

For example, a chimeric polypeptide of the disclosure comprising a GAA,laforin, alpha-amylase, AGL and/or malin polypeptide portion and aninternalizing moiety portion can be used in any of the methods of thedisclosure.

In certain embodiments, a chimeric polypeptide of the disclosure (e.g.,a polypeptide comprising a GAA, laforin, alpha-amylase, AGL and/or malinpolypeptide portion and an internalizing moiety portion) is delivered tothe cytoplasm of cells, such as muscle (e.g., skeletal muscle and/orcardiac muscle), neuronal cells and/or liver cells to decreasecytoplasmic glycogen accumulation (e.g., deleterious accumulation ofnormal of abnormal glycogen, such as polyglucosan). Such cells may bepresent in vitro or in a subject (e.g., a patient, such as a human). Incertain embodiments, the subject is a subject having, or suspected orhaving, a glycogen storage disorder, particularly Pompe Disease, GSDIII, or GSD IV, and/or a glycogen metabolism disorder, such as LaforaDisease. In certain embodiments, a chimeric polypeptide of thedisclosure is suitable for use in delivering GAA to cytoplasm in asubject in need thereof, such as a subject having Pompe Disease, GSDIII, or GSD IV, and/or a glycogen metabolism disorder, such as LaforaDisease. In certain embodiments, the subject in need thereof has or issuspected of having GSD III. In certain embodiments, the subject in needthereof has or is suspected of having GSD IV. In certain embodiments,the disclosure provides a method of treating (e.g., improving one ormore symptoms of; decreasing glycogen accumulation, such as cytoplasmicglycogen accumulation) GSD III. In certain embodiments, the disclosureprovides a method of treating (e.g., improving one or more symptoms of;decreasing glycogen accumulation, such as cytoplasmic glycogenaccumulation) GSD IV. In certain embodiments, the disclosure provides amethod of treating (e.g., improving one or more symptoms of; decreasingglycogen accumulation) Lafora Disease. Further methods are describedherein.

Without being bound by theory, although GSD III, GSD IV and LaforaDisease are not caused by mutations in GAA, both conditions arecharacterized by accumulation of glycogen. The chimeric polypeptides ofthe disclosure are suitable for delivering into cells, such as intocytoplasm of cells, to decrease glycogen accumulation (e.g., or increaseglycogen clearance). Thus, although GSD III, GSD IV and Lafora Diseaseare not caused by lack or loss of function of GAA, providing chimericpolypeptides of the disclosure may be used to treat GSD III and/or GSDIV and/or Lafora Disease, such as by decrease glycogen, such ascytoplasmic glycogen, or to improve glycogen clearance.

In some embodiments, the chimeric polypeptides of the disclosure may beused to increase glycogen clearance in a cell. In some embodiments, thecell is a muscle, liver or neuronal cell. In some embodiments, the cellis in a subject having GSDIII, GSD IV and/or Lafora Disease.

In certain embodiments, chimeric polypeptides comprising any of the GAApolypeptides disclosed herein can be used to treat any one or more ofPompe Disease, Forbes-Cori Disease, Andersen Disease, von Gierke Diseaseor Lafora Disease. In certain embodiments, chimeric polypeptidescomprising any of the AGL polypeptides disclosed herein can be used totreat Lafora Disease. In certain embodiments, chimeric polypeptidescomprising any of the malin polypeptides disclosed herein can be used totreat Lafora Disease. In certain embodiments, chimeric polypeptidescomprising any of the laforin polypeptides disclosed herein can be usedto treat Lafora Disease. In certain embodiments, chimeric polypeptidescomprising any of the alpha-amylase polypeptides disclosed herein can beused to treat Lafora Disease. In certain embodiments, chimericpolypeptides comprising any of the alpha-amylase polypeptides disclosedherein can be used to treat Forbes-Cori Disease. In certain embodiments,a subject may be treated with one or more different types of any of thechimeric polypeptides disclosed herein. For example, in someembodiments, a subject may be treated with any combination of: achimeric polypeptide comprising any of the GAA polypeptides disclosedherein, a chimeric polypeptide comprising any of the laforinpolypeptides disclosed herein, a chimeric polypeptide comprising any ofthe AGL polypeptides disclosed herein, or a chimeric polypeptidecomprising any of the malin polypeptides disclosed herein. In particularembodiments, a Lafora Disease subject is treated with at least twochimeric polypeptides selected from the group consisting of: a chimericpolypeptide comprising any of the laforin polypeptides disclosed herein,a chimeric polypeptide comprising any of the AGL polypeptides disclosedherein, a chimeric polypeptide comprising any of the alpha-amylasepolypeptides disclosed herein, and a chimeric polypeptide comprising anyof the malin polypeptides disclosed herein.

In certain embodiments, GAA polypeptides may comprise the full-lengthGAA polypeptide (e.g., a GAA polypeptide comprising the amino acidsequence of SEQ ID NO: 1 or 2). In certain embodiments, GAA polypeptidesmay comprise one of the mature, active forms of the GAA protein, such asthe 70 kDa form or the mature 76 kDa form, or a combination of the two.Mature GAA polypeptides may also be administered in combination with theimmature 110 kDa form of GAA, in order to target as many organelles andcellular regions/compartments as possible. In addition, mature GAApolypeptides may be administered in combination with and/or followingadministration of immunotolerizing fragments of GAA, such as smallfragments of GAA, and/or immunosuppressive compounds. In someembodiments, the GAA polypeptides comprise a mature GAA polypeptide aswell as additional polypeptide sequence from a GAA polypeptide, such assequence contiguous with the mature GAA polypeptide. The disclosurecontemplates that any of the chimeric polypeptides of the disclosure(e.g., a chimeric polypeptide comprising a GAA polypeptide, as describedherein and an internalizing moiety, as described here) may be used inany of the methods described herein.

In certain embodiments, the present disclosure provides methods ofdelivering chimeric polypeptides to cells, including cells in culture(in vitro or ex vivo) and cells in a subject. Delivery to cells inculture, such as healthy cells or cells from a model of disease, havenumerous uses. These uses include to identify GAA, laforin,alpha-amylase, AGL and/or malin substrates or binding partners, toevaluate localization and/or trafficking (e.g., to cytoplasm, lysosome,and/or autophagic vesicles), to evaluate enzymatic activity under avariety of conditions (e.g., pH), to assess glycogen accumulation, andthe like. In certain embodiments, chimeric polypeptides of thedisclosure can be used as reagents to understand GAA, laforin,alpha-amylase, AGL and/or malin activity, localization, and traffickingin healthy or disease contexts.

Delivery to subjects, such as to cells in a subject, has numerous uses.Exemplary therapeutic uses are described below. Moreover, the chimericpolypeptides may be used for diagnostic or research purposes. Forexample, a chimeric polypeptide of the disclosure may be detectablylabeled and administered to a subject, such as an animal model ofdisease or a patient, and used to image the chimeric polypeptide in thesubject's tissues (e.g., localization to muscle and/or liver).Additionally exemplary uses include delivery to cells in a subject, suchas to an animal model of disease (e.g., Forbes-Cori and/or AndersenDisease and/or Pompe Disease and/or von Gierke Disease and/or LaforaDisease). By way of example, chimeric polypeptides of the disclosure maybe used as reagents and delivered to animals to understand GAA, laforin,alpha-amylase, AGL and/or malin bioactivity, localization andtrafficking, protein-protein interactions, enzymatic activity, andimpacts on animal physiology in healthy or diseased animals.

In certain embodiments, the present disclosure provides methods oftreating conditions associated with, dysfunction of AGL, GAA, G6P-ase,glucose-6-phosphatase transporter, laforin, alpha-amylase, malin and/orGBE enzyme, with aberrant glycogen accumulation and/or with Forbes-Cori,Pompe Disease, von Gierke Disease, Lafora Disease and/or AndersenDisease. In certain embodiments, the glycogen accumulation is in thecytoplasms, and delivery of GAA, laforin, alpha-amylase, AGL and/ormalin reduces cytoplasmic glycogen accumulation, such as in skeletalmuscle or liver. In certain embodiments, the subject does not havedysfunction in endogenous GAA, laforin, alpha-amylase, AGL and/or malin(e.g., the methods do not comprise replacement of the protein that ismutated or for which there is dysfunction).

These methods involve, in certain embodiments, administering to theindividual a therapeutically effective amount of a chimeric polypeptideas described above (e.g., a chimeric polypeptide comprising (i) a GAAportion comprising a GAA polypeptide and (ii) an internalizing moietyportion). In certain embodiments, these methods involve administering tothe individual a therapeutically effective amount of a chimericpolypeptide as described above (e.g., a chimeric polypeptide comprising(i) a laforin polypeptide and (ii) an internalizing moiety portion). Incertain embodiments, these methods involve administering to theindividual a therapeutically effective amount of a chimeric polypeptideas described above (e.g., a chimeric polypeptide comprising (i) an AGLpolypeptide and (ii) an internalizing moiety portion). In certainembodiments, these methods involve administering to the individual atherapeutically effective amount of a chimeric polypeptide as describedabove (e.g., a chimeric polypeptide comprising (i) a malin polypeptideand (ii) an internalizing moiety portion). In certain embodiments, thesemethods involve administering to the individual a therapeuticallyeffective amount of a chimeric polypeptide as described above (e.g., achimeric polypeptide comprising (i) an alpha-amylase polypeptide and(ii) an internalizing moiety portion). These methods are particularlyaimed at therapeutic and prophylactic treatments of animals, and moreparticularly, humans. With respect to methods for treating Forbes-Coriand/or Andersen Disease and/or Pompe Disease and/or von Gierke Diseaseand/or Lafora Disease, the disclosure contemplates all combinations ofany of the foregoing aspects and embodiments, as well as combinationswith any of the embodiments set forth in the detailed description andexamples. Accordingly, chimeric polypeptides of the disclosure are, incertain embodiments, suitable for treating multiple different GSDs, suchas GSD III, and/or GSD IV, and/or Pompe Disease, and/or GSD I (includingGSD Ia and/or GSD lb and/or diseases such as Lafora Disease. In certainembodiments, the chimeric polypeptide decrease glycogen accumulation incells, such as skeletal muscle and/or liver cells, to treat GSD III,and/or GSD IV, and/or Lafora Disease, such as in a patient in need. Incertain embodiments, the same chimeric polypeptide may be used to treatmore than one GSD, such as GSD III and GSD IV. In certain embodiments,the chimeric polypeptides of the disclosure may be used to treat PompeDisease and/or von Gierke Disease (GSD1a and/or GSD1b). In certainembodiments, the chimeric polypeptide decreases glycogen accumulation incells, such as neuronal cells, to treat Lafora Disease in a patient inneed thereof.

The present disclosure provides a method of delivering a chimericpolypeptide or nucleic acid construct into a cell via an equilibrativenucleoside transporter (ENT2) pathway, comprising contacting a cell witha chimeric polypeptide or nucleic acid construct. In certainembodiments, the method comprises contacting a cell with a chimericpolypeptide, which chimeric polypeptide comprises a laforin,alpha-amylase, AGL, malin and/or a mature GAA polypeptide or bioactivefragment thereof and an internalizing moiety which can mediate transportacross a cellular membrane via an ENT2 pathway (and optionally viaanother ENT transporter, such as ENT3), thereby delivering the chimericpolypeptide into the cell. In certain embodiments, the cell is a musclecell. The muscle cells targeted using any of the methods disclosedherein may include skeletal, cardiac or smooth muscle cells. In otherembodiments, the chimeric polypeptides are delivered to liver orneuronal cells.

The present disclosure also provides a method of delivering a chimericpolypeptide or nucleic acid construct into a cell via a pathway thatallows access to cells other than muscle cells. Other cell types thatcould be targeted using any of the methods disclosed herein include, forexample, liver cells, neurons, epithelial cells, uterine cells, andkidney cells.

In certain embodiments, the internalizing moiety is an antibody orantigen binding fragment, such as an antibody or antigen bindingfragment that binds DNA. In certain embodiments, the internalizingmoiety is an antibody, such as a full length antibody or a Fab. Incertain embodiments, the full length antibody or Fab comprises one ormore substitutions, relative to a native immunoglobulin constant region,such as to decrease effector function.

Forbes-Cori Disease, also known as Glycogen Storage Disease Type III,GSD III, or limit dextrinosis, is an autosomal recessiveneuromuscular/hepatic disease with an estimated incidence of 1 in83,000-100,000 live births. Forbes-Cori Disease represents approximately24% of all Glycogen Storage Disorders. The clinical picture inForbes-Cori Disease is reasonably well established but variable.Forbes-Cori Disease patients may suffer from skeletal myopathy,cardiomyopathy, cirrhosis of the liver, hepatomegaly, hypoglycemia,short stature, dyslipidemia, slight mental retardation, facialabnormalities, and/or increased risk of osteoporosis (Ozen et al., 2007,World J Gastroenterol, 13(18): 2545-46). Forms of Forbes-Cori Diseasewith muscle involvement may present muscle weakness, fatigue and muscleatrophy. Progressive muscle weakness and distal muscle wastingfrequently become disabling as the patients enter the third or fourthdecade of life, although this condition has been reported to begin inchildhood in many Japanese patients.

Andersen Disease, also known as Glycogen Storage Disease Type IV or GSDIV, is also an autosomal recessive neuromuscular/hepatic disease with anestimated incidence of 1 in 600,000 to 800,000 individuals worldwide.The age of onset ranges from fetus to adulthood and is divided into fourgroups: (i) perinatal, presenting as fetal akinesia deformation sequenceand perinatal death; (ii) congenital, with hydrops fetalis, neuronalinvolvement and death in early infancy; (iii) childhood, with myopathyor cardiomyopathy; and (iv) adult, with isolated myopathy or adultpolyglucosan body disease (Lee, et al., 2010). Absence of enzymeactivity is usually lethal in utero or in infancy, affecting primarilymuscle and liver. However, residual enzyme activity (5-20%) leads to ajuvenile or adult-onset disorder that affects primarily muscle and bothcentral and peripheral nervous systems. Symptoms observed in AndersenDisease patients include liver dysfunction, arthrogryposis, neuronaldysfunction, failure to thrive, cirrhosis, portal vein hypertension,esophageal varices, ascites, hepatosplenomegaly, portal hypertension,hypotonia, myopathy, dilated cardiomyopathy, and shortened lifeexpectancy. These symptoms may vary in severity depending on the type ofAndersen Disease affecting the subject.

Glycogen storage disease type I (GSD I) or von Gierke Disease, is themost common of the glycogen storage diseases with an incidence ofapproximately 1 in 50,000 to 100,000-births. The deficiency impairs theability of the liver to produce free glucose from glycogen and fromgluconeogenesis, causes severe hypoglycemia and results in increasedglycogen storage in liver and kidneys. This can lead to enlargement ofboth organs.

The most common forms of GSD I are designated GSD1a and GSD1b, theformer accounting for over 80% of diagnosed cases and the latter forless than 20%. A few rarer forms have been described. GSD1a results frommutations of G6PC, the gene for glucose-6-phosphatase. GSD1b resultsfrom mutations of the SLC37A4, the glucose-6-phosphatase transporter. Incertain embodiments, patients in need of treatment with the subjectmethods are patient having GSD Ia. In other embodiments, patients inneed of treatment are patients having GSD1b.

Clinical manifestations in von Gierke Disease result, directly orindirectly, from: the inability to maintain an adequate blood glucoselevel during the post-absorptive hours of each day; organ changes due toglycogen accumulation; excessive lactic acid generation; and damage totissue from hyperuricemia. Glycogen accumulation includes accumulationin the liver and in the kidneys and small intestines. Hepatomegaly,usually without splenomegaly, begins to develop in fetal life and isusually noticeable in the first few months of life. By the time thechild is standing and walking, the hepatomegaly may be severe enough tocause the abdomen to protrude.

The kidneys of von Gierke Disease patients are usually 10 to 20%enlarged with stored glycogen. This does not usually cause clinicalproblems in childhood, with the occasional exception of a Fanconisyndrome with multiple derangements of renal tubular reabsorption,including proximal renal tubular acidosis with bicarbonate and phosphatewasting. However, prolonged hyperuricemia can cause uric acidnephropathy. In adults with GSD I, chronic glomerular damage similar todiabetic nephropathy may lead to renal failure.

Hepatic complications have been serious in some von Gierke Diseasepatients. Adenomas of the liver can develop in the second decade orlater, with a small chance of later malignant transformation to hepatomaor hepatic carcinomas. Additional problems reported in adolescents andadults with GSD I have included hyperuricemic gout, pancreatitis, andchronic renal failure.

Lafora Disease, also called Lafora progressive myoclonic epilepsy orMELF, is a rare, fatal neurodegenerative disorder characterized by theaccumulation of cytoplasmic polyglucosan inclusion bodies in cells frommost tissues of affected individuals, including the brain, heart, liver,muscle and skin. Lafora Disease patients typically first developsymptoms in adolescence. Symptoms include temporary blindness,depression, seizures, drop attacks, myoclonus, visual hallucinations,absences, ataxia and quickly developing and severe dementia. Deathusually occurs 2-10 years (5 years mean) after onset.

The prevalence of Lafora Disease is unknown. While this disease occursworldwide, it is most common in Mediterranean countries, parts ofCentral Asia, India, Pakistan, North Africa and the Middle East. InWestern countries, the prevalence is estimated to be below 1/1,000,000.

There is currently no cure or effective treatment for patients havingLafora Disease. However, the seizures and myoclonus can be managed, atleast in early stages of the disease, with antiepileptic medications.

The terms “treatment”, “treating”, and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. “Treating” a condition or disease refers to curing as well asameliorating at least one symptom of the condition or disease, andincludes administration of a composition which reduces the frequency of,or delays the onset of, symptoms of a medical condition in a subject inneed relative to a subject which does not receive the composition.“Treatment” as used herein covers any treatment of a disease orcondition of a mammal, particularly a human, and includes: (a)preventing symptoms of the disease or condition from occurring in asubject which may be predisposed to the disease or condition but has notyet begun experiencing symptoms; (b) inhibiting the disease or condition(e.g., arresting its development); or (c) relieving the disease orcondition (e.g., causing regression of the disease or condition,providing improvement in one or more symptoms). For example, “treatment”of Forbes-Cori, Pompe Disease and/or Andersen Disease encompasses acomplete reversal or cure of the disease, or any range of improvement insymptoms and/or adverse effects attributable to Forbes-Cori, PompeDisease and/or Andersen Disease. Similarly, treatment of Lafora Diseaseand/or von Gierke Disease, including GSD1a and GSD1b, is contemplatedand similarly encompasses a complete reversal or cure of the disease, orany range of improvement in symptoms and/or adverse effects attributableto the disease.

Merely to illustrate, “treatment” of Forbes-Cori Disease includes animprovement in any of the following effects associated with Forbes-CoriDisease or combination thereof: skeletal myopathy, cardiomyopathy,cirrhosis of the liver, hepatomegaly, hypoglycemia, short stature,dyslipidemia, failure to thrive, mental retardation, facialabnormalities, osteoporosis, muscle weakness, fatigue and muscleatrophy. Treatment may also include one or more of reduction of abnormallevels of cytoplasmic glycogen, decrease in elevated levels of one ormore of alanine transaminase, aspartate transaminase, alkalinephosphatase, or creatine phosphokinase, such as decrease in such levelsin serum. Improvements in any of these conditions can be readilyassessed according to standard methods and techniques known in the art.Other symptoms not listed above may also be monitored in order todetermine the effectiveness of treating Forbes-Cori Disease. Thepopulation of subjects treated by the method of the disclosure includessubjects suffering from the undesirable condition or disease, as well assubjects at risk for development of the condition or disease.

Merely to illustrate, “treatment” of Andersen Disease includes animprovement in any of the following effects associated with AndersenDisease or combination thereof: liver dysfunction, arthrogryposis,neuronal dysfunction, failure to thrive, cirrhosis, portal veinhypertension, esophageal varices, ascites, hepatosplenomegaly, portalhypertension, hypotonia, myopathy, dilated cardiomyopathy, and shortenedlife expectancy. Treatment may also include one or more of reduction ofabnormal levels of cytoplasmic glycogen. Other symptoms not listed abovemay also be monitored in order to determine the effectiveness oftreating Andersen Disease. The population of subjects treated by themethod of the disclosure includes subjects suffering from theundesirable condition or disease, as well as subjects at risk fordevelopment of the condition or disease.

In certain embodiments, the subjects in need of treatment are subjectshaving the perinatal form of Andersen Disease (e.g., perinatal form ofGSD IV). In other embodiments, the subjects in need of treatment aresubjects having the congenital (infantile) form of Andersen Disease. Inother embodiments, the subjects in need of treatment are subjects havingthe childhood (juvenile) form of Andersen Disease. In some embodiments,the subjects in need thereof are subjects having the adult form ofAndersen Disease. Thus, in certain embodiments, the disclosure providesmethods of treating any of the foregoing patients by administering achimeric polypeptide of the disclosure. In certain embodiments, thedisclosure provides methods of decreasing cytoplasmic glycogenaccumulation, such as in skeletal muscle, cardiac muscle, and/or liver,in any of the foregoing subjects in need by administering a chimericpolypeptide of the disclosure.

Merely to illustrate, “treatment” of Pompe Disease includes animprovement in any of the following effects associated with dysfunctionof GAA (or combination thereof): decreased GAA activity (e.g., treatmentincreases GAA activity), glycogen accumulation in cells (e.g., treatmentdecreases glycogen accumulation), increased creatine kinase levels,elevation of urinary glucose tetrasaccharide, heart size, hypertrophiccardiomyopathy, respiratory complications, dependence on a ventilator,muscle dysfunction and/or weakening, loss of motor function, dependenceon a wheelchair or other form of mobility assistance, dependence on neckor abdominal support for sitting upright, ultrastructural damage ofmuscle fibers, loss of muscle tone and function. Improvements in any ofthese symptoms can be readily assessed according to standard methods andtechniques known in the art. Other symptoms not listed above may also bemonitored in order to determine the effectiveness of treating PompeDisease.

In certain embodiments, the subjects in need of treatment are subjectshaving infantile form of Pompe Disease. In other embodiments, thesubjects in need of treatment are subjects having juvenile onset oradult onset Pompe Disease. Thus, in certain embodiments, the disclosureprovides methods of treating any of the foregoing patients byadministering a chimeric polypeptide of the disclosure. In certainembodiments, the disclosure provides methods of decreasing cytoplasmicglycogen accumulation, such as in skeletal muscle, cardiac muscle,and/or liver, in any of the foregoing subjects in need by administeringa chimeric polypeptide of the disclosure.

Merely to illustrate, “treatment” of von Gierke Disease includes animprovement in any of the following effects associated with von GierkeDisease or combination thereof: constant hunger, easy bruising andnosebleeds, fatigue, irritability, puffy cheeks, thin chest and limbs,swollen belly, delayed puberty, enlarged liver, gout, inflammatory boweldisease, kidney disease, kidney failure, osteoporosis, seizures,lethargy, short height, ulcers of mouth, ulcers of the bowel, livertumors, hypoglycemia, arthritis, stunted growth, pulmonary hypertension,and/or failure to grow. Other symptoms not listed above may also bemonitored in order to determine the effectiveness of treating von GierkeDisease. The population of subjects treated by the method of thedisclosure includes subjects suffering from the undesirable condition ordisease, as well as subjects at risk for development of the condition ordisease. In certain embodiments, the subject being treated is anadolescent and is treated before the onset of puberty.

Merely to illustrate, “treatment” of Lafora Disease includes animprovement in any of the following effects associated with LaforaDisease or combination thereof: blindness, depression, seizures, dropattacks, hepatic disease, muscle atrophy, myoclonus, visualhallucinations, absences, ataxia, dementia, and/or shortened lifespan.Other symptoms not listed above may also be monitored in order todetermine the effectiveness of treating Lafora Disease. The populationof subjects treated by the method of the disclosure includes subjectssuffering from the undesirable condition or disease, as well as subjectsat risk for development of the condition or disease. In certainembodiments, the subject being treated is treated before onset ofdementia or before onset of measureable, appreciable dementia.

In certain embodiments, the disclosure provides methods of deliveringGAA, laforin, alpha-amylase, AGL and/or malin activity to cells, such asmuscle and/or liver and/or kidney cells of a subject having Forbes CoriDisease, Andersen Disease, Pompe Disease, von Gierke Disease or LaforaDisease comprising administering a chimeric polypeptide of thedisclosure or a composition comprising a chimeric polypeptide of thedisclosure formulated with one or more pharmaceutically acceptablecarriers and/or excipients.

By the term “therapeutically effective dose” is meant a dose thatproduces the desired effect for which it is administered. The exact dosewill depend on the purpose of the treatment, and will be ascertainableby one skilled in the art using known techniques (see, e.g., Lloyd(1999) The Art, Science and Technology of Pharmaceutical Compounding).

In certain embodiments, administration of a chimeric polypeptide of thedisclosure is via any one of the routes of administration describedherein, such as subcutaneous, intravenous, or via the hepatic portalvein. In other words, the disclosure contemplates methods of delivery byadministering via any such route of administration. In certainembodiments, the method results in delivery of greater GAA, laforin,alpha-amylase, AGL and/or malin activity to the cytoplasm, incomparison, to that following deliver of a GAA, laforin, alpha-amylase,AGL and/or malin polypeptide that is not conjugated to an internalizingmoiety and/or in comparison to that of a GAA, laforin, alpha-amylase,AGL and/or malin polypeptide conjugated to a different internalizingmoiety.

In certain embodiments, one or more chimeric polypeptides of the presentdisclosure can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, chimeric polypeptides ofthe present disclosure can be administered alone or in combination withone or more additional compounds or therapies for treating Pompe Diseaseand/or Forbes-Cori Disease and/or von Gierke Disease and/or LaforaDisease and/or Andersen Disease. For example, one or more chimericpolypeptides can be co-administered in conjunction with one or moreother therapeutic compounds. In some embodiments, the one or morechimeric polypeptides can be co-administered in conjunction withalglucosidase alfa (Myozyme, Genzyme Corporation). Whenco-administration is indicated, the combination therapy may encompasssimultaneous or alternating administration. In addition, the combinationmay encompass acute or chronic administration. Optionally, the chimericpolypeptide of the present disclosure and additional compounds act in anadditive or synergistic manner for treating Forbes-Cori and/or AndersenDisease and/or Pompe Disease and/or von Gierke Disease and/or LaforaDisease. Additional compounds to be used in combination therapiesinclude, but are not limited to, small molecules, polypeptides,antibodies, antisense oligonucleotides, and siRNA molecules. Dependingon the nature of the combinatory therapy, administration of the chimericpolypeptides of the disclosure may be continued while the other therapyis being administered and/or thereafter. Administration of the chimericpolypeptides may be made in a single dose, or in multiple doses. In someinstances, administration of the chimeric polypeptides is commenced atleast several days prior to the other therapy, while in other instances,administration is begun either immediately before or at the time of theadministration of the other therapy.

One type of combination therapy makes use of molecules that promotemuscle synthesis and/or fat reduction. Molecules such as IGF-1, growthhormones, steroids, (3-2 agonists (for example Clenbuterol), andmyostatin inhibitors may be administered to patients in order to buildmuscle tissue and reduce fat infiltration. These molecules may alsoincrease ENT2 levels. Accordingly, the molecules may be administeredbefore treatment with a chimeric polypeptide of the disclosure begins,in between treatments, or after treatment with a chimeric polypeptide ofthe disclosure.

In some embodiments, any of the chimeric polypeptides described hereinare administered to a subject (e.g., a subject having Lafora Disease) incombination with an anti-epileptic drug.

In another example of combination therapy, one or more chimericpolypeptides of the disclosure can be used as part of a therapeuticregimen combined with one or more additional treatment modalities. Byway of example, such other treatment modalities include, but are notlimited to, dietary therapy, occupational therapy, physical therapy,ventilator supportive therapy, massage, acupuncture, acupressure,mobility aids, assistance animals, and the like.

In certain embodiments, one or more chimeric polypeptides of the presentdisclosure can be administered prior to or following a liver transplant.

Note that although the chimeric polypeptides described herein can beused in combination with other therapies, in certain embodiments, achimeric polypeptide is provided as the sole form of therapy. Regardlessof whether administrated alone or in combination with other medicationsor therapeutic regiments, the dosage, frequency, route ofadministration, and timing of administration of the chimericpolypeptides is determined by a physician based on the condition andneeds of the patient. The disclosure contemplates that a method maycomprise administration at a dose and on a dosing schedule, such asadministration at specified intervals over a period of time. In suchcases, each dose contributes to efficacy, and is thus effective,although improvement in symptoms may only be observed afteradministration of multiple doses.

Chimeric polypeptides of the disclosure have numerous uses, including invitro and in vivo uses. In vivo uses include not only therapeutic usesbut also diagnostic and research uses in, for example, any of theforegoing animal models. By way of example, chimeric polypeptides of thedisclosure may be used as research reagents and delivered to animals tounderstand GAA bioactivity, localization and trafficking,protein-protein interactions, enzymatic activity, and impacts on animalphysiology in healthy or diseases animals.

Chimeric polypeptides may also be used in vitro to evaluate, forexample, GAA, laforin, alpha-amylase, AGL and/or malin bioactivity,localization and trafficking, protein-protein interactions, andenzymatic activity in cells in culture, including healthy and GAA,laforin, alpha-amylase, AGL and/or malin deficient cells in culture. Thedisclosure contemplates that chimeric polypeptides of the disclosure maybe used to deliver GAA, laforin, alpha-amylase, AGL and/or malin tocytoplasm, lysosome, and/or autophagic vesicles of cells, includingcells in culture.

The disclosure contemplates that any of the methods described herein maybe carried out by administering or contacting cells with a chimericpolypeptide of the disclosure and/or a composition of the disclosure(e.g., a composition comprising a chimeric polypeptide of the disclosureformulated with one or more pharmaceutically acceptable carriers and/orexcipients).

VI. Gene Therapy

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding polypeptides of GAA (e.g., matureGAA), laforin, alpha-amylase, AGL and/or malin and or chimericpolypeptides comprising GAA, laforin, alpha-amylase, AGL and/or malin inmammalian cells or target tissues. In certain embodiments, the chimericpolypeptides for use in the methods described herein comprise a GAApolypeptide, but also include additional polypeptide sequence from a GAApolypeptide, including sequence contiguous with the GAA polypeptide.Such methods can be used to administer nucleic acids encodingpolypeptides of the disclosure (e.g., GAA, laforin, alpha-amylase, AGLand/or malin including variants thereof, and include chimericpolypeptides) to cells in vitro. The disclosure contemplates that genetransfer methods may be used to deliver nucleic acid encoding any of thechimeric polypeptides of the disclosure or GAA, laforin, alpha-amylase,AGL and/or malin polypeptides. In some embodiments, the nucleic acidsencoding GAA, laforin, alpha-amylase, AGL and/or malin are administeredfor in vivo or ex vivo gene therapy uses. In other embodiments, genedelivery techniques are used to study the activity of chimericpolypeptides or GAA, laforin, alpha-amylase, AGL and/or malinpolypeptide or to study Forbes-Cori and/or Andersen Disease and/or PompeDisease and/or von Gierke Disease and/or Lafora Disease in cell based oranimal models, such as to evaluate cell trafficking, enzyme activity,and protein-protein interactions following delivery to healthy ordiseased cells and tissues. Non-viral vector delivery systems includeDNA plasmids, naked nucleic acid, and nucleic acid complexed with adelivery vehicle such as a liposome. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. Such methods are well known in theart.

Methods of non-viral delivery of nucleic acids encoding engineeredpolypeptides of the disclosure include lipofection, microinjection,biolistics, virosomes, liposomes, immunoliposomes, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection methods and lipofectionreagents are well known in the art (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids that are suitable for efficientreceptor-recognition lipofection of polynucleotides include those ofFeigner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivoadministration) or target tissues (in vivo administration). Thepreparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art.

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding GAA (e.g., mature GAA), laforin, alpha-amylase, AGLand/or malin or its variants take advantage of highly evolved processesfor targeting a virus to specific cells in the body and trafficking theviral payload to the nucleus. Viral vectors can be administered directlyto patients (in vivo) or they can be used to treat cells in vitro andthe modified cells are administered to patients (ex vivo). Conventionalviral based systems for the delivery of polypeptides of the disclosurecould include retroviral, lentivirus, adenoviral, adeno-associated andherpes simplex virus vectors for gene transfer. Viral vectors arecurrently the most efficient and versatile method of gene transfer intarget cells and tissues. Integration in the host genome is possiblewith the retrovirus, lentivirus, and adeno-associated virus genetransfer methods, often resulting in long term expression of theinserted transgene. Additionally, high transduction efficiencies havebeen observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SW), human immuno deficiency virus(HIV), and combinations thereof, all of which are well known in the art.

In applications where transient expression of the polypeptides of thedisclosure is preferred, adenoviral based systems are typically used.Adenoviral based vectors are capable of very high transductionefficiency in many cell types and do not require cell division. Withsuch vectors, high titer and levels of expression have been obtained.This vector can be produced in large quantities in a relatively simplesystem. Adeno-associated virus (“AAV”) vectors are also used totransduce cells with target nucleic acids, e.g., in the in vitroproduction of nucleic acids and peptides, and for in vivo and ex vivogene therapy procedures. Construction of recombinant AAV vectors aredescribed in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al.; Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.

Replication-deficient recombinant adenoviral vectors (Ad) can beengineered such that a transgene replaces the Ad E1a, E1b, and E3 genes;subsequently the replication defector vector is propagated in human 293cells that supply deleted gene function in trans. Ad vectors cantransduce multiple types of tissues in vivo, including nondividing,differentiated cells such as those found in the liver, kidney and musclesystem tissues. Conventional Ad vectors have a large carrying capacity.

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and 42 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by producer cell linethat packages a nucleic acid vector into a viral particle. The vectorstypically contain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the protein to be expressed. The missingviral functions are supplied in trans by the packaging cell line. Forexample, AAV vectors used in gene therapy typically only possess ITRsequences from the AAV genome which are required for packaging andintegration into the host genome. Viral DNA is packaged in a cell line,which contains a helper plasmid encoding the other AAV genes, namely repand cap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. A viral vector is typically modified to have specificityfor a given cell type by expressing a ligand as a fusion protein with aviral coat protein on the viruses outer surface. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. This principle can be extended to other pairs of virusexpressing a ligand fusion protein and target cell expressing areceptor. For example, filamentous phage can be engineered to displayantibody fragments (e.g., FAB or Fv) having specific binding affinityfor virtually any chosen cellular receptor. Although the abovedescription applies primarily to viral vectors, the same principles canbe applied to nonviral vectors. Such vectors can be engineered tocontain specific uptake sequences thought to favor uptake by specifictarget cells, such as muscle cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, by systemic administration (e.g., intravenous,intraperitoneal, intramuscular, subdermal, or intracranial infusion) ortopical application. Alternatively, vectors can be delivered to cells exvivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. For example, cells areisolated from the subject organism, transfected with a nucleic acid(gene or cDNA) encoding, e.g., GAA (e.g., mature GAA), laforin,alpha-amylase, AGL and/or malin or its variants, and re-infused backinto the subject organism (e.g., patient). Various cell types suitablefor ex vivo transfection are well known to those of skill in the art.

In certain embodiments, stem cells are used in ex vivo procedures forcell transfection and gene therapy. The advantage to using stem cells isthat they can be differentiated into other cell types in vitro, or canbe introduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Stem cells are isolated fortransduction and differentiation using known methods.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can be also administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent disclosure, as described herein.

VII. Methods of Administration

Various delivery systems are known and can be used to administer thechimeric polypeptides of the disclosure. Any such methods may be used toadminister any of the chimeric polypeptides described herein. Thedisclosure contemplates than any of the methods of administrationdisclosed herein may be used to deliver any of the chimeric polypeptidesof the disclosure in the context of any of the methods described herein(e.g., methods of treatment; methods of reducing cytoplasmic glycogenaccumulation).

Methods of introduction can be enteral or parenteral, including but notlimited to, intradermal, intramuscular, intraperitoneal,intramyocardial, intravenous, subcutaneous, pulmonary, intranasal,intraocular, epidural, intrathecal, intracranial, intraventricular andoral routes. The chimeric polypeptides may be administered by anyconvenient route, for example, by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local.

In certain embodiments, the chimeric polypeptide is administeredintravenously.

In certain embodiments, it may be desirable to administer the chimericpolypeptides of the disclosure locally to the area in need of treatment(e.g., muscle); this may be achieved, for example, and not by way oflimitation, by local infusion during surgery, by means of a catheter, orby means of an implant, the implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,fibers, or commercial skin substitutes.

In another embodiment, such local administration can be to all or aportion of the heart. For example, administration can be byintrapericardial or intramyocardial administration. Similarly,administration to cardiac tissue can be achieved using a catheter, wire,and the like intended for delivery of agents to various regions of theheart.

In another embodiment, local administration is directed to the liver.Glycogen storage and glycogenolysis in the liver affect the availabilityof glycogen for many other tissues in the body. For example, a venouscatheter may be placed in the hepatic portal vein to deliver chimericpolypeptides directly to the liver. In addition, in some embodimentswhere the internalizing moieties of the chimeric polypeptides show alower affinity for liver cells than for other cell types, deliverythrough the hepatic portal vein ensures that adequate concentrations ofGAA (e.g., mature GAA), laforin, alpha-amylase, AGL and/or malin reachthe liver cells.

Note that the disclosure contemplates methods in which chimericpolypeptides are administered, at the same or different times, via onethan one route of administration. For example, the disclosurecontemplates a regimen in which chimeric polypeptides are administeredsystemically, such as by intravenous infusion, in combination with localadministration via the hepatic portal vein.

In other embodiments, the chimeric polypeptides of the disclosure can bedelivered in a vesicle, in particular, a liposome (see Langer, 1990,Science 249:1527-1533). In yet another embodiment, the chimericpolypeptides of the disclosure can be delivered in a controlled releasesystem. In another embodiment, a pump may be used (see Langer, 1990,supra). In another embodiment, polymeric materials can be used (seeHoward et al., 1989, J. Neurosurg. 71:105). In certain specificembodiments, the chimeric polypeptides of the disclosure can bedelivered intravenously.

In certain embodiments, the chimeric polypeptides are administered byintravenous infusion. In certain embodiments, the chimeric polypeptidesare infused over a period of at least 10, at least 15, at least 20, orat least 30 minutes. In other embodiments, the chimeric polypeptides areinfused over a period of at least 60, 90, or 120 minutes. Regardless ofthe infusion period, the disclosure contemplates that each infusion ispart of an overall treatment plan where chimeric polypeptide isadministered according to a regular schedule (e.g., weekly, monthly,etc.).

The foregoing applies to any of the chimeric polypeptides, compositions,and methods described herein. The disclosure specifically contemplatesany combination of the features of such chimeric polypeptides,compositions, and methods (alone or in combination) with the featuresdescribed for the various pharmaceutical compositions and route ofadministration described in this section.

VIII. Pharmaceutical Compositions

In certain embodiments, the subject chimeric polypeptides for use in anyof the methods disclosed herein are formulated with a pharmaceuticallyacceptable carrier (e.g., formulated with one or more pharmaceuticallyacceptable carriers and/or excipients). One or more chimericpolypeptides can be administered alone or as a component of apharmaceutical formulation (composition). Any of the chimericpolypeptides described herein may be formulated, as described herein,and any such compositions (e.g., pharmaceutical compositions, orpreparations, or formulations) may be used in any of the methodsdescribed herein. In certain embodiments, the composition comprises achimeric polypeptide comprising a full-length GAA polypeptide (e.g., aGAA polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or2). In other embodiments, the composition comprises a chimericpolypeptide comprising a mature GAA polypeptide. In certain embodiments,the composition includes two or more chimeric polypeptides of thedisclosure, such as a chimeric polypeptide comprising a mature GAA ofapproximately 70 kDa and a chimeric polypeptide comprising a mature GAAof approximately 76 kDa. In other embodiments, the composition comprisesa chimeric polypeptide comprising a laforin polypeptide. In otherembodiments, the composition comprises a chimeric polypeptide comprisingan AGL polypeptide. In other embodiments, the composition comprises achimeric polypeptide comprising a malin polypeptide. In otherembodiments, the composition comprises a chimeric polypeptide comprisingan alpha-amylase polypeptide. The chimeric polypeptides may beformulated for administration in any convenient way for use in human orveterinary medicine. Wetting agents, emulsifiers and lubricants, such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, release agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe compositions.

Formulations of the subject chimeric polypeptides include, for example,those suitable for oral, nasal, topical, parenteral, rectal, and/orintravaginal administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated and the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.

In certain embodiments, methods of preparing these formulations orcompositions include combining another type of therapeutic agents and acarrier and, optionally, one or more accessory ingredients. In general,the formulations can be prepared with a liquid carrier, or a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct.

In certain embodiments, any of the pharmaceutical compositions describedherein comprise concentrated amounts of any of the chimeric polypeptidesdescribed herein. In some embodiments, the compositions have 50%, 100%,150%, 200%, 250%, 300%, 350% or 400% more concentrated levels of thechimeric polypeptide as compared to the levels of chimeric polypeptideoriginally purified from the cells producing the chimeric polypeptide.

In some embodiments, the concentration of the chimeric polypeptide is atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 mg/ml. In some embodiments, the concentration of the chimericpolypeptide is at least 10 mg/ml or greater. In some embodiments, theconcentration of the chimeric polypeptide is at least 15 mg/ml orgreater. In some embodiments, the concentration of the chimericpolypeptide is at least 20 mg/ml or greater. In some embodiments, theconcentration of the chimeric polypeptide is at least 30 mg/ml orgreater. In some embodiments, the concentration of the chimericpolypeptide is at least 50 mg/ml or greater. In some embodiments, theconcentration of the chimeric polypeptide is at least 70 mg/ml orgreater. In some embodiments, the concentration of the chimericpolypeptide is at least 90 mg/ml or greater. In some embodiments, theconcentration of the chimeric polypeptide is at least 110 mg/ml orgreater. In some embodiments, the concentration of the chimericpolypeptide is 10-50 mg/ml, 10-40 mg/ml, 10-30 mg/ml, 10-25 mg/ml, 10-20mg/ml. 20-50 mg/ml, 50-70 mg/ml, 70-90 mg/ml or 90-110 mg/ml. In someembodiments, any of the compositions described herein preserve at least80%, 90%, 95% or 100% biological activity (as defined herein) for atleast 24 hours, 2 days, 4 days, 1 week, 2 weeks or 1 month when storedin a pharmaceutically acceptable formulation at 4° C. In someembodiments of any of the foregoing, the chimeric polypeptide portion ofthe composition is substantially pure, as described herein (e.g.,greater than 85% of the GAA present is in association or interconnectedwith an internalizing moiety).

Formulations for oral administration may be in the form of capsules,cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), powders, granules, or as a solutionor a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a subject chimeric polypeptide therapeuticagent as an active ingredient. Suspensions, in addition to the activecompounds, may contain suspending agents such as ethoxylated isostearylalcohols, polyoxyethylene sorbitol, and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, and mixtures thereof.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more chimericpolypeptide therapeutic agents of the present disclosure may be mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose, and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like. Liquiddosage forms for oral administration include pharmaceutically acceptableemulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the active ingredient, the liquid dosage forms maycontain inert diluents commonly used in the art, such as water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor, and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, coloring,perfuming, and preservative agents. In certain embodiments, methods ofthe disclosure include topical administration, either to skin or tomucosal membranes such as those on the cervix and vagina. The topicalformulations may further include one or more of the wide variety ofagents known to be effective as skin or stratum corneum penetrationenhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone,dimethylacetamide, dimethylformamide, propylene glycol, methyl orisopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents mayfurther be included to make the formulation cosmetically acceptable.Examples of these are fats, waxes, oils, dyes, fragrances,preservatives, stabilizers, and surface active agents. Keratolyticagents such as those known in the art may also be included. Examples aresalicylic acid and sulfur. Dosage forms for the topical or transdermaladministration include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and inhalants. The subjectpolypeptide therapeutic agents may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers (e.g., HEPES buffer), or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to a subjectchimeric polypeptide agent, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof. Powders and sprays can contain, in additionto a subject chimeric polypeptides, excipients such as lactose, talc,silicic acid, aluminum hydroxide, calcium silicates, and polyamidepowder, or mixtures of these substances. Sprays can additionally containcustomary propellants, such as chlorofluorohydrocarbons and volatileunsubstituted hydrocarbons, such as butane and propane.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more chimeric polypeptides in combination with one ormore pharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers(e.g., HEPES buffer), bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the disclosure include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

These compositions may also contain adjuvants, such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

Injectable depot forms are made by forming microencapsule matrices ofone or more polypeptide therapeutic agents in biodegradable polymerssuch as polylactide-polyglycolide. Depending on the ratio of drug topolymer, and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

In a preferred embodiment, the chimeric polypeptides of the presentdisclosure are formulated in accordance with routine procedures as apharmaceutical composition adapted for intravenous administration tohuman beings. Where necessary, the composition may also include asolubilizing agent and a local anesthetic such as lidocaine to ease painat the site of the injection. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

In another embodiment, the chimeric polypeptides of the presentdisclosure are formulated for subcutaneous administration to humanbeings.

In certain embodiments, the chimeric polypeptides of the presentdisclosure are formulated for intrathecal, intracranial and/orintraventricular delivery. In certain embodiments, a chimericpolypeptide of the disclosure for use in treating Lafora Disease or foruse in decreasing glycogen accumulation in neurons, such as in a subjecthaving Lafora Disease, is formulated for intrathecal, intracranialand/or intraventricular delivery. In certain embodiments, a method ofthe disclosure, such as a method of treating Lafora Disease or fordecreasing glycogen accumulation in neurons comprising delivering achimeric polypeptide of the disclosure intrathecally, intracraniallyand/or intraventricularly.

In certain embodiments, the chimeric polypeptides of the presentdisclosure are formulated for deliver to the heart, such as forintramyocardial or intrapericaridal delivery.

In certain embodiments, the composition is intended for localadministration to the liver via the hepatic portal vein, and thechimeric polypeptides are formulated accordingly.

Note that, in certain embodiments, a particular formulation is suitablefor use in the context of deliver via more than one route. Thus, forexample, a formulation suitable for intravenous infusion may also besuitable for delivery via the hepatic portal vein. However, in otherembodiments, a formulation is suitable for use in the context of oneroute of delivery, but is not suitable for use in the context of asecond route of delivery.

The amount of the chimeric polypeptides of the disclosure which will beeffective in the treatment of a tissue-related condition or disease(e.g., Pompe Disease and/or Forbes-Cori and/or Andersen Disease and/orvon Gierke Disease and/or Lafora Disease) can be determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the condition, and should bedecided according to the judgment of the practitioner and each subject'scircumstances. However, suitable dosage ranges for intravenousadministration are generally about 20-5000 micrograms of the activechimeric polypeptide per kilogram body weight. Suitable dosage rangesfor intranasal administration are generally about 0.01 pg/kg body weightto 1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

In certain embodiments, compositions of the disclosure, includingpharmaceutical preparations, are non-pyrogenic. In other words, incertain embodiments, the compositions are substantially pyrogen free. Inone embodiment the formulations of the disclosure are pyrogen-freeformulations which are substantially free of endotoxins and/or relatedpyrogenic substances. Endotoxins include toxins that are confined insidea microorganism and are released only when the microorganisms are brokendown or die. Pyrogenic substances also include fever-inducing,thermostable substances (glycoproteins) from the outer membrane ofbacteria and other microorganisms. Both of these substances can causefever, hypotension and shock if administered to humans. Due to thepotential harmful effects, even low amounts of endotoxins must beremoved from intravenously administered pharmaceutical drug solutions.The Food & Drug Administration (“FDA”) has set an upper limit of 5endotoxin units (EU) per dose per kilogram body weight in a single onehour period for intravenous drug applications (The United StatesPharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). Whentherapeutic proteins are administered in relatively large dosages and/orover an extended period of time (e.g., such as for the patient's entirelife), even small amounts of harmful and dangerous endotoxin could bedangerous. In certain specific embodiments, the endotoxin and pyrogenlevels in the composition are less then 10 EU/mg, or less then 5 EU/mg,or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg,or less then 0.001 EU/mg.

In some embodiments, the disclosure provides a composition, such as apharmaceutical composition comprising a chimeric polypeptide of thedisclosure formulated with one or more pharmaceutically acceptablecarriers and/or excipients. Such compositions include compositionscomprising any of the internalizing moiety portions, described herein,and a GAA, laforin, alpha-amylase, AGL and/or malin portion comprising,as described herein. For example, the disclosure provides compositionscomprising a GAA-containing chimeric polypeptide, an AGL-containingchimeric polypeptide, a laforin-containing chimeric polypeptide, analpha-amylase-containing chimeric polypeptide or a malin-containingchimeric polypeptide. In certain embodiments, any of the compositionsdescribed herein may be described based on any of the GAA, laforin,alpha-amylase, AGL and/or malin portions and/or internalizing moietyportions described herein. Moreover, any such compositions may bedescribed based on any of the structural and/or functional featuresdescribed herein. Any such compositions may be used in any of themethods described herein, such as administered to cells and/or tosubjects in need of treatment, such as administered to cells and/or tosubjects having Pompe Disease, von Gierke Disease, Forbes Cori Disease,Lafora Disease or Andersen Disease. Any such compositions may be used todeliver GAA, laforin, alpha-amylase, AGL and/or malin activity intocells, such as into muscle and/or liver cells in a patient in needthereof (e.g., a patient having Pompe Disease, von Gierke Disease,Forbes Cori Disease, Lafora Disease or Andersen Disease).

In certain embodiments, the disclosure provides compositions comprisinga GAA-containing chimeric polypeptide, and the GAA present in acomposition is enriched such that a substantial percentage of the GAApresent in the composition is the same or substantially the same, suchas has substantially the same amino acid sequence or the sameinterconnection to an internalizing moiety. For example, the presence inthe chimeric polypeptide of all or a portion of an immunoglobulin or anepitope tag, such as an HA or myc tag, provides a region forpurification of chimeric polypeptide. In some embodiments, a tag or theimmunoglobulin portion of the chimeric polypeptide are used forpurification such that a composition comprising a chimeric polypeptideof the disclosure is enriched and or substantially purified relative toGAA portions that are not interconnected to an internalizing moietyportion. For example, in certain embodiments, the presence of GAA isenriched such that greater than 90% (greater than 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or greater than 99%) of the GAA in acomposition is presented as a polypeptide interconnected to aninternalizing moiety. In other embodiments, the composition is enrichedsuch that greater than 80%, greater than 85%, greater than 90% orgreater than 95% (e.g., greater than 80%, 85%, 87%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or greater than 99%) of the GAA in acomposition is approximately the same molecular weight and/or differs atthe N-terminus of the GAA portion by less than 5, 4, 3, 2, or 1residues. In other words, in certain embodiments, less than 20% (e.g,less than 10%, 9%, 8%, 7%, 6%, 5%) of the GAA present in the compositionhas a differ molecular weight and/or differs at the N-terminus of theGAA portion by less than 5, 4, 3, 2 or 1 residue and/or is notinterconnected to an internalizing moiety.

Such compositions, including any of the compositions described herein,may be provided, for example, in a bottle or syringe and stored prior toadministration.

In certain embodiments, the disclosure provides for a pharmaceuticalcomposition comprising any of the chimeric polypeptides describedherein, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% of the GAA polypeptide in the composition are interconnectedto an internalizing moiety. In some embodiments, the pharmaceuticalcomposition comprises chimeric polypeptides wherein at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the chimericpolypeptides in the composition have the identical amino acid sequence,or an amino acid sequence that differs by less than 10, 9, 8, 7, 6, 5,4, 3, 2, or 1 amino acid residues. In some embodiments, thepharmaceutical composition comprises chimeric polypeptides wherein atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of theGAA polypeptides in the composition have the identical amino acidsequence, or an amino acid sequence that differs by less than 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 amino acid residues. In some embodiments, thepharmaceutical composition comprises chimeric polypeptides wherein atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of thechimeric polypeptides or the GAA polypeptides in the composition havethe same or substantially the same molecular weight. In certainembodiments, the composition is substantially free of mature GAA thatdoes not include additional contiguous GAA sequence.

In certain embodiments, the disclosure provides a composition comprisinga chimeric polypeptide formulated with one or more pharmaceuticallyacceptable carriers and/or excipients, which chimeric polypeptidecomprises (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising mature GAA) and (ii) an internalizing moiety thatpromotes transport into cells. In certain embodiments, at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%of the GAA polypeptide present in the composition is interconnected toan internalizing moiety. In certain embodiments, such a percentage iscalculated based on the GAA species in the composition, as evaluated bySEC or coomasie stained gel. In other words, in certain embodiments, atleast 85% of the GAA species present in the composition, as evaluated bySEC or coomasie stained gel is interconnected to an internalizingmoiety, such as associated with an antibody or Fab. In otherembodiments, such a percentage is by weight (e.g., at least 85% byweight of the GAA polypeptide present in the composition isinterconnected to an internalizing moiety, such as associated with anantibody or Fab.

In certain embodiments, the disclosure provides a composition comprisinga chimeric polypeptide formulated with one or more pharmaceuticallyacceptable carriers and/or excipients, which chimeric polypeptidecomprises (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising mature GAA) and (ii) an internalizing moiety thatpromotes transport into cells. In certain embodiments, greater than 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, orgreater than 99% of the GAA present in the composition has substantiallythe same amino acid sequence. In certain embodiments, greater than 90%(91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than 99%) of the GAApolypeptide present in the composition has the same interconnection toan internalizing moiety. In certain embodiments, at least 95% of the GAApolypeptide present in the composition is interconnected to aninternalizing moiety.

In certain embodiments, greater than 85% (greater than or at least 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greaterthan or at least 99%) of the GAA polypeptide present in the compositionis approximately the same molecular weight.

In certain embodiments, greater than 90% (greater than or at least 91%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greaterthan or at least 99%) of the GAA polypeptide present in the compositiondiffers at the N-terminus of a GAA polypeptide portion by less than 5,4, 3, 2, or 1 residues. In certain embodiments, greater than 85%(greater than or at least 86%, 87%, 88%, 89%, 90%, 91%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than or at least99%) of the GAA polypeptide present in the composition differs at theC-terminus of a GAA polypeptide portion by less than 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 residues.

In certain embodiments, the composition is substantially free of matureGAA that does not include additional contiguous GAA sequence and/or thatis not interconnected to an internalizing moiety. In certainembodiments, the composition is substantially free of mature GAA that isnot interconnected to an internalizing moiety.

In certain embodiments, the composition is substantially free of matureGAA. In certain embodiments, the composition comprises less than 5%,such as by weight, of mature GAA that does not include additionalcontiguous GAA sequence and/or that is not interconnected to aninternalizing moiety.

In certain embodiments, the disclosure provides a composition comprisingchimeric polypeptides formulated with one or more pharmaceuticallyacceptable carriers and/or excipients, which chimeric polypeptidescomprise (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising mature GAA) and (ii) an internalizing moiety thatpromotes transport into cells. In certain embodiments, at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or atleast 99% of the chimeric polypeptides in the composition comprise anamino acid sequence that differs by less than 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 amino acid residues.

In certain embodiments, the disclosure provides a composition comprisingchimeric polypeptides formulated with one or more pharmaceuticallyacceptable carriers and/or excipients, which chimeric polypeptidescomprise (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising mature GAA) and (ii) an internalizing moiety thatpromotes transport into cells. In certain embodiments, at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or atleast 99% of the GAA present in the composition comprises an amino acidsequence that differs by less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid residues.

In certain embodiments, the disclosure provides a composition comprisinga chimeric polypeptide formulated with one or more pharmaceuticallyacceptable carriers and/or excipients, which chimeric polypeptidecomprises (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising mature GAA) and (ii) an internalizing moiety thatpromotes transport into cells. In certain embodiments, the compositionis substantially free of mature GAA that does not include additionalcontiguous GAA sequence and/or that is not interconnected to aninternalizing moiety.

In certain embodiments, the disclosure provides a composition comprisingchimeric polypeptides formulated with one or more pharmaceuticallyacceptable carriers and/or excipients, which chimeric polypeptidescomprise (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising mature GAA) and (ii) an internalizing moiety thatpromotes transport into cytoplasm of cells. In certain embodiments, atleast 91% (greater than 90% or at least 91%, or greater than or at least92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than or at least 99%) ofthe GAA polypeptide present in the composition is interconnected to aninternalizing moiety. In certain embodiments, this percentage isdetermined under conditions that preserve the associate of an antibodyheavy and light chain.

In certain embodiments, the disclosure provides a composition comprisinga chimeric polypeptide formulated with one or more pharmaceuticallyacceptable carriers and/or excipients, which chimeric polypeptidecomprises (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising) and (ii) an internalizing moiety that promotestransport into cells. In certain embodiments, at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%of the chimeric polypeptides present in the composition have the sameamino acid sequence.

In certain embodiments, the disclosure provides a composition comprisinga chimeric polypeptide formulated with one or more pharmaceuticallyacceptable carriers and/or excipients, which chimeric polypeptidecomprises (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising mature GAA) and (ii) an internalizing moiety thatpromotes transport into cells. In certain embodiments, at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or atleast 99% of the GAA present in the composition has the same amino acidsequence.

In certain embodiments, the disclosure provides a composition comprisinga chimeric polypeptide formulated with one or more pharmaceuticallyacceptable carriers and/or excipients, which chimeric polypeptidecomprises (i) an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAApolypeptide comprising mature GAA) and (ii) an internalizing moiety thatpromotes transport into cells. In certain embodiments, less than 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the GAA present inthe composition is a mature GAA polypeptide. In certain embodiments,less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of theGAA present in the composition is a mature GAA polypeptide that is notassociated or interconnected with an internalizing moiety.

In certain embodiments, including certain embodiments of any of theforegoing or other embodiments, a percentage is calculated based on theGAA species (or the chimeric polypeptide species) in the composition, asevaluated by SEC or coomasie stained gel. In other words, in certainembodiments, at least 85% of the GAA species (or chimeric polypeptidespecies) present in the composition, as evaluated by SEC (e.g.,SEC-HPLC) or coomasie stained gel, is interconnected to an internalizingmoiety, such as associated with an antibody or Fab, or at least 85% ofthe GAA species (such as species conjugated to an internalizing moiety)or chimeric polypeptide species present in the composition have the sameamino acid sequence or an amino acid sequence that differs by less thanfor example, 10, 9, 8, 7, 6, or 5 amino acid residues. In otherembodiments, such a percentage is by weight (e.g., at least 85% byweight of the GAA polypeptide present in the composition isinterconnected to an internalizing moiety, such as associated with anantibody or Fab). In certain embodiments, SEC or coomasie blue stainingis performed under conditions that maintain the association of the heavyand light chain of an antibody or antigen binding fragment, such as whenthe internalizing moiety is an antibody or antigen binding fragment.

In certain embodiments, at least 85%, at least 90%, at least 91%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% of the polypeptide in the formulation is a chimericpolypeptide comprising a GAA polypeptide associated or interconnectedwith an internalizing moiety. In certain embodiments, the percentage ofpolypeptide in the composition is by weight and/or accessed by SEC orcoomasie blue staining.

In certain embodiments, any of the foregoing percentages (e.g., at least85% or greater than 91%) may also be expressed as a range (e.g.,85%-95%, 90-98%, 91-95%, 91-96%, 91-97%, 91-98%, 91-99%, 95-97%, 95-98%,95-99%, etc.).

In certain embodiments of any of the foregoing, the GAA polypeptideportion is any of the GAA polypeptide portions described herein and theinternalizing moiety portion is any of the internalizing moiety portionsdescribed herein.

The foregoing applies to any of the chimeric polypeptides, compositions,and methods described herein. The disclosure specifically contemplatesany combination of the features of such chimeric polypeptides,compositions, and methods (alone or in combination) with the featuresdescribed for the various pharmaceutical compositions and route ofadministration described in this section.

IX. Animal Models

a. Forbes-Cori Disease

Curly-coated retriever dogs having a frame-shift mutation in their AGLgene display a disease similar to Forbes-Cori Disease in humans (Yi, etal., 2012, Disease Models and Mechanisms, 5: 804-811). These dogspossess abnormally high glycogen deposits in their liver and muscle,and, consistent with muscle and liver damage, possess high and graduallyincreasing levels of alanine transaminase, aspartate transaminase,alkaline phosphatase and creatine phosphokinase in their serum. See, Yiet al. In addition these dogs displayed progressive liver fibrosis anddisruption of muscle cell contractile apparatus and the fraying ofmyofibrils. See, Yi et al. As such, this canine model of Forbes-Coriclosely resembles the human disease, with glycogen accumulation in liverand skeletal muscle that leads to progressive hepatic fibrosis andmyopathy. See, Yi et al.

A mouse model of Forbes-Cori also has recently been developed. In thismodel, mice possess a single ENU-induced base pair mutation within theAGL gene. Similar to human patients of Forbes-Cori, these mice exhibitpersistently elevated levels of alanine transaminase and aspartatetransaminase, which levels are indicative of liver damage.

Anstee, et al., 2011, J. Hepatology, 54(Supp 1-Abstract 887): S353.These mice also display markedly increased hepatic glycogen deposition.See, Anstee et al. As such, these mice display several key features alsoobserved in human patients of Forbes-Cori Disease.

These models provide suitable animal model systems for assessing theactivity and effectiveness of the subject chimeric polypeptides. Thesemodels have correlation with symptoms of Forbes-Cori Disease, and thusprovide an appropriate model for studying Forbes-Cori Disease. Activityof the polypeptide can be assessed in one or both models, and theresults compared to that observed in wildtype control animals andanimals not treated with the chimeric polypeptides. Assays that may beused for assessing the efficacy of any of the chimeric polypeptidesdisclosed herein in treating the Forbes-Cori mouse or dog include, forexample: assays assessing alanine transaminase, aspartate transaminase,alkaline phosphatase and/or creatine phosphokinase levels in the serum;assessing glycogen levels in a biopsy from the treated and untreatedForbes-Cori mice or dogs (e.g., by examining glycogen levels in a muscleor liver biopsy using, for example, periodic acid Schiff staining fordetermining glycogen levels); assessing tissue glycogen levels (See,e.g., Yi et al., 2012); and/or monitoring muscle function, cardiacfunction, liver function, and/or lifespan in the treated and untreatedForbes-Cori dogs or mice. Another example of an in vitro assay fortesting activity of the chimeric polypeptides disclosed herein would bea cell or cell-free assay in which whether the ability of the chimericpolypeptides to hydrolyze 4-methylumbelliferyl-α-D-glucoside as asubstrate is assessed.

b. Andersen Disease

Norwegian Forest Cats that are homozygous for a loss of exon 12 in theirGBE1 gene display a disease similar to Andersen Disease in humans (Fyfe,et al., 2007, Molecular Genetics and Metabolism, 90(4): 383-392. Themajority of cats harboring this disease died shortly after birth.Surviving Andersen Disease cats would appear normal until approximately5 months of age before severe muscular weakness, atrophy, contracturesand inability to use the hind limbs would result. The cells of manytissues, including muscle cells, hepatocytes and neurons, in these catswere characterized by having clusters of inclusion bodies that stainpositive for glycogen (Fyfe et al., 1992, Pediatric Research, 32(6):719-725). Several tissues, including skeletal muscle, cardiac muscle andneurons of the central nervous system, showed signs of degeneration. Thecats that survived to adulthood often died suddenly from heart failure(Fyfe et al, 1992).

Several mouse models of Andersen Disease have also been developed. Anearly onset Andersen Disease mouse model was developed by utilizing aFLPe-mediated homozygous deletion of exon 7 (Akman, 2011, Hum Mol Genet,20(22):4430-9 and Akman, 2014, Neurology, 82(1):P1.054). Mice lackingexon 7 had no GBE activity, and an early onset lethality. Another earlyonset, fetal Andersen Disease model has been generated, in which micewere engineered to carry a stop mutation (E609X) in the Gbel gene usinga gene-driven ENU (N-ethyl-N-nitrosurea)-mutagenesis approach. TheseE609X mice display hydrops fetalis and lethality between mid- and late-gestation, recapitulating the clinical features of severe fetalneuromuscular forms of human Andersen Disease (Lee, et al., 2010, HumMol Genet, 20(3):455-465). In addition, juvenile and adult onset modelsof Andersen Disease have been developed. For example, a juvenile andadult onset mouse model of Andersen Disease was generated that containsa kinase-neomycin cassette within intron 7 of the GBE gene, resulting indecreased GBE expression. This juvenile and adult onset mouse modeldisplays progressive neuromuscular dysfunction, aberrant glycogenaccumulation in muscle cells and hepatocytes, and shortened lifespan(Akman, et al., 2011). Another adult onset Andersen Disease model wasgenerated in which the Y329S human mutation was inserted into exon 7 ofthe mouse Gbel gene, resulting in reduced GBE activity in these mice(Akman, 2014). Transgenic mice homozygous for the Y329S mutation exhibita phenotype similar to adult onset Andersen Disease, with widespreadaccumulation of polyglucosan. These Y329S mice also display progressiveneuromuscular dysfunction.

c. von Gierke Disease

Mice engineered to be deficient in either G6Pase-a or G6PT activity werefound to mimic human cases of GSD-Ia and GSD-Ib, respectively (Lei etal., 1996, Nat Genet., 13:203-209; Chen et al., 2003, Hum Mol Genet,12:2547-2558; Kim et al., 2007, FEBS Lett., 581(20):3833-38). GSD-Ibmice manifest metabolic abnormalities characteristic of disturbedglucose homeostasis and also suffer from neutropenia and neutrophildysfunctions characteristic of GSD-Ib. Similar to human cases of GSD-Ia,the GSD-Ia mice have markedly increased levels of granulocyte colonystimulating factor (G-CSF) and cytokine-induced neutrophilchemoattractant (KC).

A canine model of GSD-Ia also exists (Kishnani et al., 2001, Vet Pathol,38(1):83-91) and is similar clinically, biochemically and pathologicallyto human cases of GSD-Ia. The canine model is homozygous for the M121IGSD-Ia mutation, which results in a mutated, defective G6P-ase gene.Dogs homozygous for this mutation exhibit tremors, weakness andneurologic signs when hypoglycemic. In addition, these animals hadpostnatal growth retardation and progressive hepatomegaly. Biochemicalabnormalities were observed in these animals, including fastinghypoglycemia, hyperlactacidemia, hypercholesterolemia,hypertriglyceridemia, and hyperuricemia. In the kidneys of some of thediseased animals, there was segmental glomerular sclerosis andvacuolation of proximal convoluted tubular epithelium. These animals arealso associated with increased liver glycogen content and isolatedmarkedly reduced G-6-Pase enzyme activity in liver and kidney (Kishnaniet al., 2001).

d. Lafora Disease

Mice engineered to be deficient in malin display a phenotype similar tothat observed in human cases of Lafora Disease. Specifically,malin^(−/−) mice presented in an age-dependent manner neurodegeneration,increased synaptic excitability, and propensity to suffer myoclonicseizures. Valles-Ortega et al., 2011, EMBO Mol Med, 3(11):667-681. Inaddition, these mice accumulated glycogen-filled inclusion bodies thatwere most abundant in the hippocampus and cerebellum, but that were alsofound in skeletal and cardiac muscle cells. Valles-Ortega et al.Glycogen was also found to be less branched in the cells of malin^(−/−)mice as compared to glycogen observed in the cells of healthy controlmice. Valles-Ortega et al. An increased level of glycogenhyperphosphorylation has also been described in this mouse model.Turnbull et al., 2010, Ann Neurol, 68(6):925-33.

Mice engineered to be deficient in laforin also display some phenotypicsimilarities to human cases of Lafora Disease. Specifically,laforin^(−/−) mice are born developmentally normal, but develop anage-dependent ataxia and myoclonus epilepsy. Ganesh et al., 2002, HumMol Genet, 11(11):1251-62. In addition, laforin^(−/−) mice displaywidespread degeneration of neurons by two months of age and thedevelopment of inclusion bodies by 4-12 months of age. Ganesh et al.,2002. Mice deficient for laforin also display hyperphosphorylation andaggregation of tau protein in the brain. Puri et al., 2009, J Biol Chem,284(34):22657-63.

e. Pompe Disease

Pompe Disease has been modeled in animals such as Brahman and Shorthorncattle, Lapland dog, cats, sheep, and a strain of Japanese quail(Kikuchi et al., Clinical and Metabolic Correction of Pompe Disease byEnzyme Therapy in Acid Maltase-deficient Quail, J. Clin. Invest.,101(4): 827-833, 1998). In addition, mouse models have been developed bytargeted disruption of the GAA gene (summarized in Geel et al., PompeDisease: Current state of treatment modalities and animal models,Molecular Genetics and Metabolism, 92:299-307, 2007). Briefly, micepossessing a knockout in exon 13 of the GAA gene exhibit glycogenaccumulation in lysosomes of liver, heart, and skeletal muscle cells,but remain phenotypically normal (Bijvoet et al., Generalized glycogenstorage and cardiomegaly in a knockout mouse model of Pompe Disease,Human Molecular Genetics, 7(1): 53-62, 1998). Mice in which exon 6 ofthe GAA gene was replaced by a neomycin resistance gene flanked by LoxPsites was developed, and lacked GAA function in several tissues. Thismouse has also been crossed with Cre-producing mice, and the resultantprogeny have abnormal lysosomal glycogen storage in heart and skeletalmuscle (Raben et al., Targeted Disruption of the Acid a-Glucosidase Genein Mice Causes an Illness with Critical Features of Both Infantile andAdult Human Glycogen Storage Disease Type II, J. Biological Chemistry,272(30): 19086-19092, 1998). A similar mouse model has targetedreplacement of exon 14 with a neomycin cassette and is comparable to theneomycin-exon 6 mouse (Raben et al., Modulation of disease severity inmice with targeted disruption of the acid alpha-glucosidase gene,Neuromuscl. Disord. 10: 283-291, 2000). Two additional mouse models havebeen developed to address issues of immune response: one mouse model inwhich the exon 6 deletion was targeted to maintain GAA function in theliver while keeping the disease phenotype in other tissues, and one GAAknockout mouse model in SCID mice, which do not produce anti-hGAAantibodies upon administration of hGAA (Raben et al., Induction oftolerance to a recombinant human enzyme, acid alpha-glucosidase, inenzyme deficient knockout mice, Transgenic Research, 12:171-178, 2003;Xu et al., Improved efficacy of gene therapy approaches for PompeDisease using a new, immune-deficient GSD-II mouse model, Gene Therapy,11:15890-1598, 2004). More recently, a double KO mouse has beendeveloped that pairs deletion of GAA and deletion of glycogen synthase 1to help determine the effects of decreased glycogen production (Xu etal., Impaired organization and function of myofilaments in single musclefibers from a mouse model of Pompe Disease, J Appl Physiol 108:1383-1388, 2010).

f. Forbes-Cori Disease and Andersen Disease and von Gierke Disease andLafora Disease and Pompe Disease

Accordingly, in certain embodiments, the present disclosure contemplatesmethods of surveying improvements in disease phenotypes using the GAAconstructs of the disclosure (e.g., the chimeric polypeptides comprisingmature GAA of the disclosure, such as chimeric polypeptides comprising aGAA polypeptide portion and an internalizing moiety portion) disclosedherein in any one or more animal models, such as the mouse modelsdescribed herein. By way of example, various parameters can be examinedin experimental animals treated with a subject chimeric polypeptide, andsuch animals can be compared to controls. Exemplary parameters that canbe assessed to evaluate potential efficacy include, but are not limitedto: increase in lifespan; increase in glycogen clearance, decrease inglycogen accumulation, and improved muscle strength, for example in openfield and open wire hang paradigms, improved heart function, improvedliver function, improved kidney function, or decrease in liver size.Increase in glycogen clearance and decrease in glycogen accumulation maybe assessed, for example, by periodic acid Schiff staining in a biopsy(e.g., muscle, liver or neuronal) from a treated or untreated animalmodel. In certain embodiments, the disclosure provides a method ofdecreasing cytoplasmic glycogen accumulation in a subject having any ofthe foregoing conditions.

Moreover, a complete pharmacokinetic study to determine the effectivedose, clearance rate, volume of distribution, and half-life of any ofthe chimeric polypeptides described herein is determined. The PK/PD/TKof the final product can be examined in larger animals such as rats,dogs, and primates.

The above models are exemplary of suitable animal model systems forassessing the activity and effectiveness of the subject chimericpolypeptides and/or formulations. These models have correlations withsymptoms of GSDI, GSD II, GSD III, GSD IV, and Lafora Disease, and thusprovide appropriate models for studying von Gierke, Pompe Disease,Forbes-Cori, Andersen Disease and/or Lafora Disease, respectively.Activity of the subject chimeric polypeptides and/or formulations isassessed in any one or more of these models, and the results compared tothat observed in wildtype control animals and animals not treated withthe chimeric polypeptides (or treated with GAA, laforin, alpha-amylase,AGL and/or malin alone). Similarly, the subject chimeric polypeptidescan be evaluated using cells in culture, for example, cells preparedfrom any of the foregoing mutant mice or other animals, as well as wildtype cells, such as fibroblasts, myoblasts or hepatocytes. Cells fromsubjects having the disease may also be used. Additionally, cell freesystems may be used to assess, for example, enzymatic activity of thesubject chimeric polypeptides. An example of an in vitro assay fortesting activity of the chimeric polypeptides disclosed herein would beto treat Pompe, von Gierke, Forbes-Cori, Lafora and/or Andersen Diseasecells with or without the chimeric polypeptides and then, after a periodof incubation, stain the cells for the presence of glycogen, e.g., byusing a periodic acid Schiff (PAS) stain. Another example of an in vitroassay for testing activity of the chimeric polypeptides, e.g., achimeric polypeptide comprising a GAA polypeptide, disclosed hereinwould be a cell or cell-free assay in which the ability of the chimericpolypeptides to hydrolyze 4-methylumbelliferyl-α-D-glucoside as asubstrate is assessed. Cell proliferation, morphology and cell death mayalso be monitored in treated or untreated cells.

Chimeric polypeptides of the disclosure have numerous uses, including invitro and in vivo uses. In vivo uses include not only therapeutic usesbut also diagnostic and research uses in, for example, any of theforegoing animal models. By way of example, chimeric polypeptides of thedisclosure may be used as research reagents and delivered to animals tounderstand GAA, laforin, alpha-amylase, AGL and/or malin bioactivity,localization and trafficking, protein-protein interactions, enzymaticactivity, and impacts on animal physiology in healthy or diseasedanimals.

Chimeric polypeptides may also be used in vitro to evaluate, forexample, GAA, laforin, alpha-amylase, AGL and/or malin bioactivity,localization and trafficking, protein-protein interactions, andenzymatic activity in cells in culture, including healthy, diseased (butnot GAA, laforin, alpha-amylase, AGL and/or malin deficient) and GAA,laforin, alpha-amylase, AGL and/or malin deficient cells in culture. Thedisclosure contemplates that chimeric polypeptides of the disclosure maybe used to deliver GAA, laforin, alpha-amylase, AGL and/or malin tocytoplasm, lysosome, and/or autophagic vesicles of cells, includingcells in culture.

Chimeric polypeptide, such as GAA chimeric polypeptides, may further beused to identify protein-protein interactions in systems where a proteinsuch as GAA is not deficient, such as in Lafora Disease. Chimericpolypeptides may further be used to understand the relative benefit ofdecreasing accumulation of glycogen in certain cell types butpotentially not all cell types in which symptoms are present. Chimericpolypeptides may be used to identify substrates for GAA, laforin,alpha-amylase, AGL and/or malin particularly in settings whereendogenous GAA, laforin, alpha-amylase, AGL and/or malin is not mutated.Chimeric polypeptides are useful for evaluating trafficking of GAA,laforin, alpha-amylase, AGL and/or malin and the chimeric polypeptidesin healthy, as well as diseased cells where glycogen accumulation is dueto different underlying causes.

X. Kits

In certain embodiments, the disclosure also provides a pharmaceuticalpackage or kit comprising one or more containers filled with at leastone chimeric polypeptide of the disclosure. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects (a)approval by the agency of manufacture, use or sale for humanadministration, (b) directions for use, or both.

In certain embodiments, the kit includes additional materials tofacilitate delivery of the subject chimeric polypeptides. For example,the kit may include one or more of a catheter, tubing, infusion bag,syringe, and the like. In certain embodiments, the chimeric polypeptideis packaged in a lyophilized form, and the kit includes at least twocontainers: a container comprising the lyophilized chimeric polypeptideand a container comprising a suitable amount of water, buffer (e.g.,HEPES buffer), or other liquid suitable for reconstituting thelyophilized material.

The foregoing applies to any of the chimeric polypeptides, compositions,and methods described herein. The disclosure specifically contemplatesany combination of the features of such chimeric polypeptides,compositions, and methods (alone or in combination) with the featuresdescribed for the various kits described in this section.

EXEMPLIFICATION

The disclosure now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present disclosure, and are not intended to limit the disclosure.For example, the particular constructs and experimental design disclosedherein represent exemplary tools and methods for validating properfunction. As such, it will be readily apparent that any of the disclosedspecific constructs and experimental plan can be substituted within thescope of the present disclosure.

Example 1 Generation of 3E10 mAb-GAA and 3E10 Fab-GAA Fusion Constructs

Representative chimeric polypeptides were expressed according to theprotocol described in Hacker et al., 2013, Protein Expr Purif. 92: 67.Specifically, chimeric polypeptides comprising a GAA polypeptide portionand an internalizing moiety portion were made recombinantly. In thisexperiment, a GAA polypeptide comprising a GAA polypeptide was fused toeither a full-length murine monoclonal 3E10 antibody comprising thelight chain variable domain set forth in SEQ ID NO: 10 and the heavychain variable domain set forth in SEQ ID NO: 9 (the internalizingmoiety portion), or to a Fab of this 3E10 antibody (see FIG. 1).Specifically, in this example, a GAA polypeptide having the amino acidsequence of SEQ ID NO: 22 was fused to the C-terminus of either theheavy chain constant region of a murine 3E10 Fab fragment or to theC-terminus of the heavy chain constant region of a full-length murine3E10 monoclonal antibody (mAb). In this example, the heavy chain of theinternalizing moiety comprises murine 3E10 antibody comprises theforegoing VH and a murine heavy chain constant domain comprising CHLhinge, CH2, and CH3 regions (in the case of the full length antibody) ora heavy chain constant domain comprising a CH1 and upper hinge region,such as constant domain regions from an IgG1, IgG2a, IgG2b, or IgG4antibody. In either case, a nucleotide sequence expressing therecombinant heavy chain and a nucleotide sequence encoding a light chaincomprising the foregoing 3E10 VL were inserted into separate vectors andtransiently transfected into CHO-DG44 cells in order to produce therecombinant, chimeric protein. Similarly, the nucleotide sequenceencoding the heavy and light chains could be expressed from a singlevector. The chimeric constructs are shown schematically in FIG. 1.

In this example, a linker sequence was used to fuse the GAA polypeptideto the Fab or mAb heavy chains, and that linker had the amino acidsequence of SEQ ID NO: 30. This provides examples of chimericpolypeptides in which the internalizing moiety was a full lengthantibody, as well as chimeric polypeptides in which the internalizingmoiety was an antigen binding fragment, here a Fab. Chimericpolypeptides in which the GAA portion comprises or consists of any ofthe GAA polypeptides described herein are also contemplated and aresimilarly made, as well as chimeric polypeptides in which theinternalizing moiety portion is any of the internalizing moiety portionsdescribed herein and are similarly made, and all suitable combinations.

Example 2 Generation and Characterization of mu3E10 mAb-GAA and 3E10Fab-GAA

Chimeric polypeptides comprising a GAA polypeptide portion and aninternalizing moiety were made recombinantly. Here, the internalizingmoiety was either a full length antibody or a Fab, comprising a heavychain variable domain as set forth in SEQ ID NO: 9 and a light chainvariable domain as set forth in SEQ ID NO: 10. For both the 3E10 mAb andthe 3E10 Fab proteins, a light chain having the amino acid sequence ofSEQ ID NO: 35, which includes a signal sequence (SEQ ID NO: 33), wasused. However, it is recognized that the signal sequence is cleaved andnot present in the final protein product. For both the 3E10 mAb and the3E10 Fab proteins, a GAA polypeptide having the amino acid sequence ofSEQ ID NO: 22 was fused (e.g., the proteins were made as a fusionprotein) to the C-terminal portion of either the 3E10 mAb or 3E10 Fabheavy chain by means of a flexible gly-ser linker (SEQ ID NO: 30). The3E10 mAb heavy chain consisted of the amino acid sequence of SEQ ID NO:37 (signal sequence of SEQ ID NO: 36+VH sequence of SEQ ID NO: 9) andthe following constant domain scheme: murine IgG2a CH1-muIgG1hinge-muIgG1 CH2-CH3. The mu3E10 Fab heavy chain consisted of the aminoacid sequence of SEQ ID NO: 37 (signal sequence of SEQ ID NO: 36 +VHsequence of SEQ ID NO: 9) and the following constant domain scheme:murine IgG2a

CH1-muIgG1 upper hinge. The signal sequences of SEQ ID NO: 33 and SEQ IDNO: 36 are not present in the mature mu3E10 mAb or mu3E10 Fab proteins(e.g., the signal sequence is not present in the final antibody product,but is cleaved during production).

For both the chimeric polypeptide wherein the internalizing moiety is afull length antibody and the chimeric polypeptide wherein theinternalizing moiety is a Fab, a nucleotide sequence expressing therecombinant heavy chain and light chain were transiently transfectedinto CHOExpress™ cells in order to produce the recombinant, chimericprotein. Both the 3E10 mAb and 3E10 Fab chimeric polypeptides showedstrong expression and secretion from the transfected CHOExpress™ cells.In similar experiments, a humanized form of the 3E10 Fab-GAA fusionprotein was also generated using the CHOExpress™ cell expression system.Specifically, humanized 3E10 Fab-GAA was expressed by transient CHO cellexpression and purified on CaptureSelect IgG-CH1 Affinity Matrix. Inseparate experiments, the humanized Fab-GAA was purified on CaptureSelect CH1 Affinity Matrix followed by further purification by SPcation-exchange. Humanized Fab comprises humanized 3E10 V_(H) and V_(L)and human constant regions.

For several representative lots, purity of humanized Fab-GAA protein wasassessed using SEC-HPLC. Briefly, SEC-HPLC was performed as a puritymethod to determine main, pre- and post-peak purities by using a TosohG3000swxl, 7.8×300mm, 5 μm column and a mobile phase consisting of 0.1MCitrate, 0.1M NaCl pH 4.5. Test samples were diluted in mobile phase toa final concentration of 2 mg/mL and 10 uL was injected. The column flowrate was 0.5 mL/min and the column was held at a constant 25° C. Theeluted peaks were detected by absorbance at 280 nm. As shown in FIG. 2,humanized Fab-GAA protein was obtained at greater than 96% purity asmeasured by SEC-HPLC. In other words, humanized Fab-GAA was present atgreater than 95% by weight of the protein present in the composition.

For one lot, Fab-GAA was then formulated at a concentration of 3.56mg/ml in a buffer comprising 33 mM citrate, 150 mM NaCl, and 332 mOsm/kgat pH 4.0 and stored at −70° C. In one experiment, a sample from thislot was then concentrated to either 10 or 15 mg/ml, and assessed forstructural and functional stability over time. Briefly, multiple vialsof huFab-GAA (3.66 mg/ml) were pooled and applied to an Amicon Ultra 4mL, 10 k MWCO spin filter (Millipore Cat#UFC801096). Material wascentrifuged for approximately 20 minutes at 4000×g at 5° C. until targetconcentrations of 10 and 15 mg/ml were obtained. The sample wasconcentrated in its current buffer and no buffer exchange was performed.The target concentration was estimated by monitoring volume of retentateby comparing the weight pre and post concentration and assuming adensity of 1.0 for sample solution. Determination of proteinconcentration was by UV Spectroscopy at a wavelength of 280nm using amolar absorbance of 1.595. It was determined that concentrating Fab-GAAto 10 or 15 mg/ml did not have any observable impact on proteinstructure (as determined by SDS-PAGE analysis) and did not result in anysignificant reduction in specific activity (as measured using thecell-free activity assay described below) when compared to the structureand activity of Fab-GAA in the original lot (3.66 mg/ml). In addition,this preservation of structural integrity and enzymatic activity of theconcentrated fusion proteins was sustained for at least seven days.

In addition, humanized 3E10 Fab-GAA polypeptide was also expressed usinga retroviral expression system. Specifically, a retrovector made from agene construct developed to express a humanized form of the murine 3E10VL set forth above and a humanized form of the murine 3E10 VH set forthabove was used to express Fab-GAA protein in CHO cells.

Fab-GAA in a Cell-Free Activity Assay

Fifty micrograms of purified Fab-GAA fusion in 100 mM acetic acid (pH4.9) was buffer-exchanged into lx PBS (pH 7.4) using a zeba desaltingspin column. Fab-GAA fusion protein was incubated in PBS (pH 7.4) at 37°C. for 0, 1, 4, 12 and 24 hours and centrifuged prior to removing analiquot for use in an enzyme assay. For each time point, following theforegoing incubation in PBS, 10 μl of enzyme was pipetted into 90 μl of100 mM sodium acetate pH 4.3 and stored at −70° C. until analysis. Eachtime point sample was analyzed using a fluorometric plate-based assayusing 4-methylumbelliferyl α-D-glucosidase (MU-α-Glu) substrate. Fab-GAAactivity was found to be similar following incubation at pH 7.4 at alltime points tested. These data indicate that the Fab-GAA fusion retainsactivity for up to 24 hours at pH 7.4. Similar activity assayexperiments were performed using a humanized 3E10 Fab-fusion protein.For a representative lot of humanized Fab-GAA, GAA enzymatic activitywas determined to be 11.61 μM/min/μg.

In another experiment, the effect of glucose was tested on the enzymaticactivity of the murine Fab-GAA fusions. In this experiment, the effectsof varying concentrations of glucose (0, 1, 5 and 10 mM) and pH (pH 4.3or 6.0) on activity of Fab-GAA fusion proteins were tested using theMU-a-Glu activity assay described above. The effects of pH on thesamples were tested by incubating the samples with either 0.1 M sodiumacetate (pH 4.3) or 0.1 M sodium phosphate (pH 6.0). Ninety-fivemicroliters of the MU-a-Glu substrate and the glucose solutions (0, 1, 5and 10 mM) at either pH 4.3 or pH 6.0 were added to a 96 well half areaflat black bottom plate. The Fab-GAA protein samples were diluted 1:10with pH 4.3 or 6.0 buffer and 5uL of the diluted samples were added toeach well. Time points were taken every 30 seconds for up to an hour.The slope of the linear portion of kinetic assay was used to determineactivity. It was found that glucose inhibited Fab-GAA fusion proteinactivity in a dose dependent manner. Moreover, glucose had a strongerinhibitory effect on Fab-GAA fusion protein activity at all doses testedfor samples incubated at pH 6.0 than for the samples incubated at pH4.3. A summary of the results from these experiments is provided inTable 1 below. Percent inhibition is indicated as compared to untreatedsamples (i.e., 0 mM glucose). This experiment shows that a chimericpolypeptide comprising a GAA polypeptide and an internalizing moiety hasenzymatic activity, both at pH 4.3 and pH 6.0, and that activity ismaintained in the presence of glucose. The latter characteristic isuseful for future assay development and indicates that these chimericpolypeptides can be tested in cell-based assays in media, whileretaining activity.

TABLE 1 pH and mM Fab-GAA Conc. Glucose (mg/ml) nmol/hr/ml nmol/hr/mg %inhibition pH 4.3, 0 mM 0.099 7864.2 79436.4 Glucose pH 4.3, 1 mM 0.0996894.7 69643.4 12.3 Glucose pH 4.3, 5 mM 0.099 6600.2 66668.7 16.1Glucose pH 4.3, 10 mM 0.099 6024.8 6085.6 23.4 Glucose pH 6.0, 0 mM0.099 3395.5 34298.0 Glucose pH 6.0, 1 mM 0.099 2794.0 28222.2 17.7Glucose pH 6.0, 5 mM 0.099 2219.3 22417.2 34.6 Glucose pH 6.0, 10 mM0.099 1107.8 11189.9 67.4 Glucose

Fab-GAA in Pompe Fibroblasts

The effect of murine 3E10 Fab-GAA in cells from Pompe patients was alsoassessed. Specifically, fibroblast cells from Pompe patients weremaintained in minimum essential medium Eagle supplemented with 10% FBS,100 U penicillin/ml, and 100 g streptomycin/ml at 37° C. in a 5% CO2-airatmosphere. For treatment, Fab-GAA was added to the fresh culture mediumwith 2% BSA (Sigma) and cells were incubated for 24 hours before beingwashed 3 times with cold DPBS and then harvested. Media and cell lysatesfrom the treated cells were assessed for the presence of GAA by using ananti-human GAA antibody. As demonstrated in FIG. 3, while Fab-GAA wasdetected largely as a 150 kDa band (which corresponds to the predictedmolecular weight of the complete Fab-GAA chimeric protein) in the mediaof cells, GAA was detected in the treated cell lysates as three separate150, 95 and 70 kDa bands. The 150 kDa band corresponds to the predictedmolecular weight of the complete Fab-GAA chimeric protein, the 95 kDaband corresponds to the predicted molecular weight of the intermediateform of GAA, and the 70 kDa band corresponds to the predicted molecularweight of the mature GAA polypeptide. Without being bound by theory,these results reflect internalization of Fab-GAA into Pompe patientfibroblasts where it can be processed into mature GAA polypeptide.

Treated cells were also tested to assess GAA activity and to determineeffects of Fab-GAA on glycogen reduction. Specifically, frozen cellpellets were homogenized and sonicated in distilled water, and insolubleproteins were removed by centrifugation. The protein content of theresultant lysates was quantified via Bradford assay. GAA activity wasassessed by measurement of 4-methylumbelliferyl-a-D-glucoside cleavageat pH 4.3 using the activity assay as described above. GAA activityfollowing administration of the Fab-GAA proteins was similar to thatobserved for unconjugated recombinant human GAA alone.

Glycogen content in treated cells was determined by treatment of tissueextracts with Aspergillus niger amyloglucosidase and measurement ofglucose released. From these experiments, it was found that treatmentwith Fab-GAA protein was also capable of reducing glycogen in Pompepatient fibroblasts. In addition, this glycogen reduction was partiallysensitive to free mannose-6-phosphate (M6P). Proteins modified with M6Presidues are known to be internalized by cells and targeted to endosomesby means of the M6P receptor. Treatment of cells with free M6P would beexpected to bind M6P receptors, thereby resulting in fewer M6P receptorsbeing available to bind and internalize proteins post-translationallymodified with M6P residues.

M6P-sensitive internalization into cells of at least a portion ofFab-GAA was further assessed using L6 rat skeletal muscle cells.Specifically, L6 cells were treated with either human Fab-GAA or murineFab-GAA in the presence or absence of M6P. Following treatment, cellswere lysed and assessed for levels and banding patterns of GAA. Asdemonstrated in FIG. 4, cells treated with either the human or murineFab-GAA displayed GAA processing into the predicted intermediate andmature forms of GAA following internalization. However, when Fab-GAAcells were co-administered free M6P, the levels of overall GAA as wellas the predicted intermediate and mature forms of GAA were diminished.Similar results were also observed in C2Cl2 murine myoblast cellstreated with Fab-GAA. Without being bound by theory, these results areconsistent with internalization of Fab-GAA by M6P-independent pathway,as well as via an M6P pathway.

M6P-independent internalization of Fab-GAA was further corroborated byseparate immunuocytochemistry experiments. Specifically, Pompe cellswere grown on slides overnight and then incubated with 200 U/ml murineFab-GAA in the presence or absence of 5 mM M6P at 37° C. in 5% CO2.Following 24 hours of treatment, cells were washed 4 times with DPBSbefore fixing with 4% paraformaldehyde at room temperature for 1 hour.Slides were then permeabilized with 0.1% Triton X-100 for 15 minutes andblocked with blocking buffer (5% goat serum (16210064-thermo) in DPBS)for 30 minutes. Slides were incubated with primary rabbit anti-Lamp2antibody (ab37024) (1:500 in blocking buffer) for 1 hour and then withAlexa Fluor conjugated anti-mouse IgG (H+L) secondary antibodies(Invitrogen). These experiments demonstrated strong staining of theFab-GAA in Pompe cell cytoplasm in the presence or absence of M6P,providing further evidence that Fab-GAA enters cells in an M6P-receptorindependent manner and that, upon internalization into cells, Fab-GAA isnot restricted to M6P-receptor compartments (e.g., endosomes/lysosomes).

Without being bound by theory, the above data are consistent withFab-GAA being internalized into Pompe fibroblasts by two distinctmechanisms: a) by means of 3E10 Fab-mediated internalization, and b) bymeans of M6P-mediated internalization. In accordance with this model,the Fab-GAA that is internalized by means of the 3E10 Fab moiety wouldbe expected to be capable of clearing cytoplasmic glycogen, while thoseFab-GAA molecules internalized via the M6P receptor would be expected tobe capable of clearing endosomal glycogen. This two-pronged glycogenclearance approach would have significant therapeutic value, ascurrently available drugs for treating Pompe Disease, such as Myozyme®,are believed to predominantly target the endocytic/lysosomal pathway.Indeed, Myozyme® does not appear to treat glycogen accumulation incytoplasm (Schoser et al., Therapeutic approaches in Glycogen StorageDisease type II (GSDII)/Pompe Disease, Neurotherapeutics, 5(4): 569-578,2008).

Treatment of Pompe Mice with Fab-GAA

A Pompe mouse model (B6 129-Gaa^(tm/Rabn/J); Jackson Laboratory) hasbeen previously described, and this model recapitulates key features ofPompe Disease in humans. This GAA^(−/−) model was utilized to test thetherapeutic efficacy of a humanized

Fab-GAA fusion protein. Specifically, five 12-week-old GAA^(−/−) micewere treated with humanized Fab-GAA and five 12-week-old GAA^(−/−) miceserved as untreated controls. Over the course of a four week study,treated mice received four separate intravenous injections of 30 mg/kgFab-GAA that was normalized to achieve 7.0 μM/min activity. Mice weresacrificed 48 hours after the last injection, and tissues were collectedfor further analysis.

Tissues from treated and untreated animals were assessed for GAAactivity and glycogen content. Specifically, frozen animal tissues werehomogenized and sonicated in distilled water, and insoluble proteinswere removed by centrifugation. The protein content of the resultantlysates was quantified via the Bradford assay. GAA activity was assessedby measurement of 4-methylumbelliferyl-a-D-glucoside cleavage at pH 4.3,and was found to be dramatically increased in all tissues tested (liver,heart, diaphragm, quadriceps muscle, gastrocnemius muscle, and spleen)except kidney. Glycogen content was determined by treatment of tissueextracts with Aspergillus niger amyloglucosidase and measurement ofglucose released, as described (Amalfitano et al. 1999, Proc Natl AcadSci USA 96(16): 8861-8866 and Sun et al., 2003, Mol Ther 7(2): 193-201).GAA^(−/−) mice treated with Fab-GAA displayed reduced glycogen contentby 64% in liver, 55% in heart, 40% in diaphragm, 15% in quadriceps, and38% in gastrocnemius. Dramatic glycogen reduction in diaphragm andgastrocnemius was also demonstrated by PAS staining of tissue sectionstaken from treated and untreated animals. Western Blot confirmedinternalization of Fab-GAA into liver, heart, spleen and gastrocnemius.

Certain carbohydrates, such as hexose tetrasaccharide (Hex4), areelevated in a number of glycogen storage diseases, including PompeDisease. GAA^(−/−) mice also display elevated Hex4 levels, which can bemeasured in the urine of mice. See, e.g., WO 2009075815. GAA^(−/−) micetreated with Fab-GAA displayed a significant (p<0.01) reduction ofurinary Hex4 levels as compared to untreated controls.

Example 3 Chemical Conjugation of 3E10 and Human GAA (mAb3E10* GAA)Chemical Conjugation

In this example, ten milligrams (10 mg) of a full-length 3E10 mABcomprising a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO: 9 and a light chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 10 (e.g., such as an scFv in whichthe VH and VL domains are interconnected via a linker) are conjugatedcovalently, directly or indirectly, to a GAA polypeptide (e.g., a GAApolypeptide comprising amino acid residues 67-952 of SEQ ID NO: 1), in a1/1 or 1/2 molar ratio with the use of two different heterobifunctionalreagents, succinimidyl 3-(2-pyridyldithio) propionate and succinimidyltrans-4-(maleimidylmethyl) cyclo-hexane-1-carboxylate. This reactionmodifies the lysine residues of 3E10 into thiols and adds thiolreactivemaleimide groups to GAA (Weisbart R H, et al., J Immunol. 2000 Jun 1;

164(11): 6020-6). After deprotection, each modified protein is reactedto each other to create a stable thioether bond. Chemical conjugation isperformed, and the products are fractionated by gel filtrationchromatography. The composition of the fractions is assessed by nativeand SDS-PAGE in reducing and nonreducing environments. Fractionscontaining the greatest ratio of 3E10-GAA conjugate to free 3E10 andfree GAA are pooled and selected for use in later studies.

Similarly, conjugates are made in which an antigen binding portion of3E10 (such as a single chain Fv fragment) or a 3E10 Fab is conjugated toa GAA polypeptide (e.g., a GAA polypeptide comprising the amino acidsequence of SEQ ID NO: 22). Other exemplary conjugates includeconjugates in which the internalizing moiety is either a full length3E10 mAb, or variant thereof, or an antigen binding fragment of theforegoing. The foregoing methods can be used to make chemical conjugatesthat include any combination of GAA portions and internalizing moietyportions, and the foregoing are merely exemplary. Both N-terminal andC-terminal conjugates are made (e.g., conjugates in which the 3E10portion is N-terminal to the GAA portion and conjugates in which the3E10 portion is C-terminal to the GAA portion). Moreover, theexperimental approaches detailed herein can be used to evaluate any suchchimeric polypeptide or to compare activity amongst chimericpolypeptides.

In Vitro Assessment of Chemically Conjugated 3E10 and GAA

Any chimeric polypeptides of the disclosure comprising a GAA polypeptideportion (e.g., a GAA polypeptide comprising the amino acid sequence ofSEQ ID NO: 22) and an internalizing moiety portion are contemplated.Subject chimeric polypeptides are added, for example, to cell culturesand the extent of protein uptake, protein localization and/or

GAA enzymatic activity are determined and compared to controls.Similarly, GAA enzymatic activity can be assessed in cell free systems.We note that although, in this example, the internalizing moiety portionand GAA portion are chemically conjugated, each individual portion maybe made recombinantly (e.g., by expressing nucleotide sequence encodingthe polypeptide in a cell in culture and purifying the expressedpolypeptide).

i) Enzymatic Activity of 3E10- GAA

GAA enzymatic activity is measured by determining the rate of 3E10- GAAcatalyzed hydrolysis of a synthetic substrate,p-nitrophenyl-D-a-glucopyranoside, in 50 mM sodium acetate, 0.1% BSA, pH4.3, as described in McVie et al. (Biochemical and PharmacologicalCharacterization of Different Recombinant Acid α-GlucosidasePreparations Evaluated for the Treatment of Pompe Disease, Mol GenetMetab., 94(4): 448-455, 2008). The released chromophore, p-nitrophenol,is quantified spectrophotometrically at an alkaline pH (>10.2) at 400nm. One unit of GAA is defined as that amount of activity which resultedin the hydrolysis of 1 μmol of substrate per minute at 37° C. under theassay conditions. Duplicate experiments are performed for Fv3E10 andGAA, Fv3E10 alone, or GAA alone. As noted above, any of the experimentsdescribed herein may also be performed using full-length 3E10-GAA,Fab3E10-GAA, Fab′3E10-GAA or any humanized variants thereof. Moreover,chimeric polypeptides comprising any GAA portion and any internalizingmoiety portion may are similarly made and tested.

ii) Uptake of 3E10- GAA

Uptake of 3E10- GAA is first assessed in COS-7 cells. Previous studiesindicate that ENT2 is involved in 3E10 transport across the membrane ofCOS-7 cells (Hansen et al., J. Biol. Chem., 282: 20790-20793, 2007), anda similar strategy can be used to determine transport of the chimeric3E10- GAA across the membrane. Briefly, purified chimeric polypeptidesare prepared in PBS with 10% fetal calf serum; control buffer is PBSwith 10% fetal calf serum. 50μL of control buffer or 3E10- GAA is addedto COS-7 cells and incubated for 1 hour. The buffer is aspirated, cellsare washed, fixed in chilled 100% ethanol, and stained with either anantibody to 3E10 or to GAA.

To demonstrate that muscle cells also uptake 3E10- GAA polypeptides, thesame experiment is conducted in muscle cells. The murine cardiomyocteHL-1 cell line expresses ENT2 (Naydenova et al., Inosine andequilibrative nucleoside transporter 2 contribute to hypoxicpreconditioning in the murine cardiomyocyte HL-1 cell line, Am JPhysiol. Heart Circ. Physiol., 294(6):H2687-2692, 2008), and this cellline can be used in place of COS-7 cells in the above experiment.

It has been previously shown that 3E10 (produced by the above mentionedhybridoma) alone is capable of penetrating primary rat cortical neurons.Weisbart, et al., 2000, J. Immunology, 164:6020-6026. To demonstratethat neuronal cells also uptake 3E10- GAA polypeptides, cultures of ratcortical neurons from cerebral hemispheres of 16-day-old fetal Wistarrats may be used. Briefly, hemispheres are dissected under sterileconditions and mechanically dissociated and plated in polylysine-coated30-mm round coverslips placed in six-well plastic dishes (CorningCostar, Cambridge, MA). Cells will then be cultured for 7-10 days beforethe internalization experiments are performed.

iii.) Treatment of Forbes-Cori Cells with 3E10- GAA

Ten to 100 uM of chemically conjugated Fv3E10- GAA polypeptides, anunconjugated mixture of 3E10 and GAA, 3E10 alone, or GAA alone areapplied to semiconfluent, undifferentiated Forbes-Cori Disease orwildtype myoblasts or hepatocytes from curly-coated retrievers orhumans. The specificity of 3E10-GS3-GAA for the ENT2 transporter isvalidated by addition of nitrobenzylmercaptopurine riboside (NBMPR), anENT2 specific inhibitor (Hansen et al., 2007, J. Biol. Chem., 282(29):20790-3) to ENT2 transfected cells just prior to addition of 3E10-GAA.It is appreciated, however, that an internalizing moiety (including 3E10or a 3E10 variant) may also be able to transit cells via a differenttransporter, such as ENT3. Eight to 24 hours later the media and cellsare collected for immunoblot and RTPCR analysis. A duplicate experimentcan be applied to each of the above proteins onto Forbes-Cori Diseaseand wildtype myoblasts or hepatocytes grown on coverslips, followed byfixation and immunohistochemical detection of mAb3E10 using antibodiesagainst mouse kappa light chain (Jackson Immunoresearch) and GAA.

a) Immunoblot Detection of Cell Penetrating 3E10 and GAA

Cell pellets are resuspended in 500 ul PBS, lysed, and the supernatantsare collected for immunoblot analysis of mAb3E10 and GAA. Epitopetagging is not employed, therefore the presence of a coincidentanti-3E10 and anti-GAA immunoreactive band of ˜248 kDa (for the fulllength 3E10 +GAA, where the GAA portion has the amino acid sequence ofSEQ ID NO: 22) in 3E10*GAA treated cells versus 3E10-alone and GAA-alonecontrols constitutes successful penetration of chemically conjugated3E10*GAA. Tubulin detection is used as a loading control.

b) Immunofluorescence of Cell Penetrating 3E10 and GAA

Coverslips of treated cells are washed, fixed in 100% ethanol,rehydrated, and 3E10 and GAA are detected with anti-GAA antibodies,followed by a horseradish peroxidase conjugated secondary antibody,color development, and viewing by light microscopy.

c) Cytopathology Analysis

Without being bound by theory, although Andersen Disease and Forbes-Coriare not caused by mutations in GAA, both conditions are characterized byaccumulation of glycogen. The chimeric polypeptides of the disclosureare suitable for delivering into cells, such as into cytoplasm of cells,to decrease glycogen accumulation (e.g., or increase glycogenclearance). Thus, although Andersen Disease and Forbes-Cori are notcaused by lack or loss of function of GAA, providing chimericpolypeptides of the disclosure may be used to treat Andersen Disease andForbes-Cori, such as to decrease glycogen, such as cytoplasmic glycogen,or to improve glycogen clearance.

Coverslips of treated cells will be washed, fixed in 100% ethanol or in10% formalin, rehydrated, and glycogen will be detected using a periodicacid-Schiff (PAS) stain. Decreased PAS staining in the treated AndersenDisease and/or Forbes-Cori cells as compared to the untreated AndersenDisease and/or Forbes-Cori cells is indicative that the treatment iseffective in reducing glycogen accumulation in the cells.

iv.) Treatment of Andersen Disease Cells with 3E10- GAA

Ten to 100 uM of chemically conjugated Fv3E10- GAA polypeptides, anunconjugated mixture of 3E10 and GAA, 3E10 alone, or GAA alone areapplied to semiconfluent, undifferentiated Andersen Disease or wildtypemyoblasts or hepatocytes from Norwegian Forest cats or humans. Thespecificity of 3E10-GS3-GAA for the ENT2 transporter is validated byaddition of nitrobenzylmercaptopurine riboside (NBMPR), an ENT2 specificinhibitor (Hansen et al., 2007, J.Biol.Chem., 282(29): 20790-3) to ENT2transfected cells just prior to addition of 3E10-GAA. Eight to 24 hourslater the media and cells are collected for immunoblot and RTPCRanalysis. A duplicate experiment applies each of the above proteins ontoAndersen Disease and wildtype myoblasts or hepatocytes grown oncoverslips, followed by fixation and immunohistochemical detection ofmAb3E10 using antibodies against mouse kappa light chain (JacksonImmunoresearch) and GAA.

a) Immunoblot Detection of Cell Penetrating 3E10 and GAA

Cell pellets are resuspended in 500 ul PBS, lysed, and the supernatantsare collected for immunoblot analysis of mAb3E10 and GAA. Epitopetagging is not employed, therefore the presence of a coincidentanti-3E10 and anti-GAA immunoreactive band of -248 kDa (for the fulllength 3E10+GAA having the amino acid sequence of SEQ ID NO: 22) in3E10* GAA treated cells versus 3E10-alone and GAA-alone controlsconstitutes successful penetration of chemically conjugated 3E10*GAA.Tubulin detection is used as a loading control.

b) Immunofluorescence of Cell Penetrating 3E10 and GAA

Coverslips of treated cells are washed, fixed in 100% ethanol,rehydrated, and 3E10 and GAA are detected with anti-GAA antibodies,followed by a horseradish peroxidase conjugated secondary antibody,color development, and viewing by light microscopy.

c) Cytopathology Analysis

Coverslips of treated cells are washed, fixed in 100% ethanol or in 10%formalin, rehydrated, and glycogen are detected using a periodicacid-Schiff (PAS) stain. Decreased PAS staining in the treated AndersenDisease cells as compared to the untreated Andersen Disease cells isindicative that the treatment is effective in reducing glycogenaccumulation in the cells.

v.) Treatment of von Gierke Cells with 3E10-GAA

Ten to 100 uM of chemically conjugated Fv3E10- GAA polypeptides, anunconjugated mixture of 3E10 and GAA, 3E10 alone, or GAA alone isapplied to neutrophil cultures from GSD1b patients and healthy controlsaccording to the protocol described in Kuijpers, 2003, 101(12):5021-4and Nikolai et al., 2002, Blood, 99(2). The specificity of 3E10- GAA forthe ENT2 transporter may be validated by addition ofnitrobenzylmercaptopurine riboside (NBMPR), an ENT2 specific inhibitor(Hansen et al., 2007, J.Biol.Chem., 282(29): 20790-3) to ENT2transfected cells just prior to addition of 3E10-GAA. Eight to 24 hourslater the media and cells are collected for immunoblot and RT-PCRanalysis. In parallel experiments, cells are examined morphologicallyand stained for apoptotic and glycogen markers at 8, 16, or 24 hoursafter culturing/treatment.

In alternative experiments, neutrophils and/or fibroblasts and/orhepatocytes from mice engineered to be deficient in either G6Pase-a orG6PT activity (Lei et al., 1996, Nat Genet., 13:203-209; Chen et al.,2003, Hum Mol Genet, 12:2547-2558; Kim et al., 2007, FEBS Lett.,581(20):3833-38) are cultured and treated with or without Fv3E10- GAApolypeptides, an unconjugated mixture of 3E10 and GAA, 3E10 alone, orGAA alone. Effects of the 3E10-GAA polypeptides on glycogen levelsand/or on the survival, morphology, apoptosis, and proliferation of thecultured cells may be assessed using assays known in the art.

a) Immunoblot Detection of Cell Penetrating 3E10 and GAA

Cell pellets are resuspended in 500 ul PBS, lysed, and the supernatantsare collected for immunoblot analysis of mAb3E10 and GAA. Epitopetagging is not employed, therefore the presence of a coincidentanti-3E10 and anti-GAA immunoreactive band of ˜248 kDa (for the fulllength 3E10+GAA having the amino acid sequence of SEQ ID NO: 22) in3E10* GAA treated cells versus 3E10-alone and GAA-alone controlsconstitutes successful penetration of chemically conjugated 3E10*GAA.Tubulin detection can be used as a loading control.

b) Immunofluorescence of Cell Penetrating 3E10 and GAA

Coverslips of treated cells are washed, fixed in 100% ethanol,rehydrated, and 3E10 and GAA are detected with anti-GAA antibodies,followed by a horseradish peroxidase conjugated secondary antibody,color development, and viewing by light microscopy.

c) Cytopathology Analysis

In parallel experiments, the GSD-Ia treated and untreated cells areassessed for apoptotic morphology and/or for apoptotic markers, similarto the experiments described in Kuijpers, 2003, 101(12):5021-4 andNikolai et al., 2002, Blood, 99(2).

vi.) Treatment of Lafora Disease Cells with 3E10-GAA

Ten to 100 uM of chemically conjugated 3E10- GAA polypeptides, anunconjugated mixture of 3E10 and GAA, 3E10 alone, or GAA alone areapplied to fibroblasts from human Lafora Disease patients cultured in amanner similar to that described in Aguado et al., Hum Mol Genet,19(14):2867-76. The specificity of 3E10- GAA for the ENT2 transportermay be validated by addition of nitrobenzylmercaptopurine riboside(NBMPR), an ENT2 specific inhibitor (Hansen et al., 2007, J. Biol.Chem., 282(29): 20790-3) to ENT2 transfected cells just prior toaddition of 3E10-GAA. Eight to 24 hours later the media and cells arecollected for immunoblot and RT-PCR analysis. In parallel experiments,cells are examined morphologically and stained for apoptotic andglycogen markers at 8, 16, or 24 hours after culturing/treatment. Inalternative experiments, neutrophils from mice engineered to bedeficient in either G6Pase-a or G6PT activity (Lei et al., 1996, NatGenet., 13:203-209; Chen et al., 2003, Hum Mol Genet, 12:2547-2558; Kimet al., 2007, FEBS Lett., 581(20):3833-38) are cultured and treated withor without 3E10- GAA polypeptides, an unconjugated mixture of 3E10 andGAA, 3E10 alone, or GAA alone. Effects of the 3E10-GAA polypeptides onLafora Body formation, glycogen levels and on the survival, morphology,apoptosis, and proliferation of the cultured cells may be assessed usingassays known in the art.

a) Immunoblot Detection of Cell Penetrating 3E10 and GAA

Cell pellets are resuspended in 500 ul PBS, lysed, and the supernatantsare collected for immunoblot analysis of mAb3E10 and GAA and LC3-II (amarker for autophagy). Epitope tagging is not employed, therefore thepresence of a coincident anti-3E10 and anti-GAA immunoreactive band of-248 kDa (for the full length 3E10+GAA having the amino acid sequence ofSEQ ID NO: 22) in 3E10* GAA treated cells versus 3E10-alone andGAA-alone controls constitutes successful penetration of chemicallyconjugated 3E10*GAA. Tubulin detection are used as a loading control. Iflevels of LC3-II levels are elevated in Lafora Disease cells treatedwith 3E10-GAA as compared to untreated cells, this is indicative that animprovement in autophagic function may be occurring in the treatedcells. Overall protein degradation in the treated and untreated cellsmay also be assessed in a manner similar to that described in Aguado etal. in order to determine whether an improvement in autophagic functionis occurring in the treated cells.

b) Immunofluorescence of Cell Penetrating 3E10 and GAA

Coverslips of treated cells are washed, fixed in 100% ethanol,rehydrated, and 3E10 and GAA are detected with anti-GAA antibodies,followed by a horseradish peroxidase conjugated secondary antibody,color development, and viewing by light microscopy. In parallelexperiments, autophagic vesicles are detected in the cells using an LC3antibody in a manner similar to that described in Aguado et al. Anincrease in the amount of LC3 staining in the treated cells as comparedto the untreated control cells is indicative that an improvement inautophagic function may be occurring in the treated cells.

c) Cytopathology Analysis

In parallel experiments, the Lafora Disease treated and untreated cellsare assessed for periods of time ranging from 1, 2, 3, 4, 7 or 10 daysor more in culture and assessed for the presence or absence of LaforaBodies and/or monitored for cell survival.

Example 4 Genetic Construct of fv 3E10 and Human GAA (Fv3E10-GS3- GAA)

Mammalian expression vectors encoding a genetic fusion of Fv3E10 and aGAA polypeptide (e.g., a GAA polypeptide comprising the amino acidsequence of SEQ ID NO: 22)(fv3E10-GS3- GAA, comprising the scFv of mAb3E10 fused to GAA by, for example, the GS3 linker) is generated. Notethat in the examples, we have used “Fv3E10” to refer to an scFv of 3E10.Note that these genetic fusions are also referred to as recombinantconjugates or recombinantly produced conjugates. These are furtherexamples of chimeric polypeptides comprising a GAA polypeptide and aninternalizing moiety, here, an scFv. Other linkers may similarly beused. Further, linkerless fusions where the 3E10 moiety and the GAAmoiety are directly fused may also be used. Similarly fusions to aportion of a full length antibody or Fab may be made. As with thechemical conjugates, recombinant fusions comprising any of the chimericpolypeptides of the disclosure are contemplated. Recombinantly producedchimeric polypeptides may comprise a GAA polypeptide portion, accordingto the disclosure (e.g., a GAA polypeptide comprising the amino acidsequence of SEQ ID NO: 22) and an internalizing moiety portion,according to the disclosure.

Additional recombinantly produced conjugates comprising a GAApolypeptide (e.g., a GAA polypeptide comprising the amino acid sequenceof SEQ ID NO: 22) and an internalizing moiety is similarly made forlater testing. By way of non-limiting example: (a) GAA-GS3-3E10, (b)3E10-GS3- GAA, (c) GAA-GS3-Fv3E10, (d) GAA-3E10, (e) 3E10-GAA, (f)GAA-Fv3E10. Note that throughout the examples, the abbreviation Fv isused to refer to a single chain Fv of 3E10. Similarly, mAb 3E10 and 3E10are used interchangeably. These and other chimeric polypeptides can betested using, for example, the assays detailed herein. Furtherpolypeptides in which the chimeric polypeptides comprise a GAApolypeptide (e.g. a GAA polypeptide having the amino acid sequence ofSEQ ID NO: 1 or 2), are also contemplated and can similarly be made andtested.

Create the cDNA for Human GAA and Confirm Activity In Vitro

i) Synthesis of the cDNA for GAA

The full-length, 3.6 kb human GAA cDNA that encodes a full length,precursor form of human GAA (hGAA cDNA) may be found athttp://www.ncbi.nlm.nih.gov/sites/entrez, for example, under GenBankAccession No. NM_000152.3. This cDNA sequences and other transcriptvariants are hereby incorporated in their entirety. A portion of such ahuman cDNA sequence corresponding approximately to the region thatencodes a GAA polypeptide (e.g., a GAA polypeptide comprising the aminoacid sequence of SEQ ID NO: 22) is used herein to generate a recombinantconstruct. However, it is also contemplated that the full length cDNAcan be used.

The GAA cDNA along with flanking restriction sites that facilitatecloning into appropriate expression vectors is synthesized and sequencedby Genscript or other qualified manufacturer of gene sequences. Tomaximize expression, the GAA cDNA is codon optimized for mammalian andpichia expression. In the event that mammals or pichia prefer adifferent codon for a given amino acid, the next best candidate to unifythe preference is used. The resulting cDNA is cloned into a CMV-basedmammalian expression cassette and large scale preps of the plasmidpCMV-GAA is made using the Qiagen Mega Endo-free plasmid purificationkit. To avoid complicating immune responses to the 3E10-GAA protein,epitopes or purification tags are not, in certain embodiments, included.However, conjugates that do include such tags may also be made andtested.

ii) Transfection of Cells In Vitro

A strategy to assess the function of GAA in transfected cells isdescribed above. Ten micrograms of the plasmid pCMV (mock) or pCMV-GAAis transfected into 1) COS-7 cells, 2) HL-1 cells, 3) myofibers and/orhepatocytes from wildtype humans, mice or dogs, and 4) myofibers and/orhepatocytes from Forbes-Cori humans, mice or dogs using commerciallyavailable transfection reagents. In a parallel experiment, tenmicrograms of the plasmid pCMV (mock) or pCMV-GAA is transfected into 1)COS-7 cells, 2) HL-1 cells, 3) myofibers and/or hepatocytes fromwildtype humans, Norwegian Forest cats and/or mice, and 4) myofibersand/or hepatocytes from Andersen Disease humans, Norwegian Forest catsor mice using commercially available transfection reagents. Similarly,any of the control or diseased von Gierke and/or Lafora Disease cellsdescribed herein may be used in these transfection experiments andmonitored in the paramaters indicated in Example 2. To track theefficiency of transfection, duplicate transfections with plasmidsencoding a suitable reporter such as beta-galactosidase or GFP isperformed. Forty-eight hours later transfected cells are pelleted bycentrifugation resuspended in 500 μl PBS for protein and immunoblotanalysis.

iii) Viral Infection with AAV cDNA Construct

Constructs described above are cloned into an adenovirus vector plasmid,according to methods described in Sun et al., (Enhanced Efficacy of anAAV Vector Encoding Chimeric, Highly-Secreted Acid a-glucosidase inGlycogen Storage Disease Type II, Mol Ther., 14(6): 822-830, 2006).These constructs provide a means to test the cDNA constructs in cells,and/or use constructs in vivo for gene therapy.

Briefly, 293 cells are transfected with an AAV vector plasmid, the AAVpackaging plasmid p5E18-VD 2/8, and pAdHelper (Stratagene, La Jolla,Calif.). Cell lysate is harvested 48 hours following infection,freeze-thawed 3 times, and isolated by sucrose cushion pelletingfollowed by 2 cesium chloride gradient centrifugation steps. AAV stocksare dialyzed against 3 changes of Hanks buffer, and aliquots are storedat −80 ° C. The number of vector DNA containing-particles is determinedby DNase I digestion, DNA extraction, and Southern blot analysis. Allviral vector stocks are handled according to Biohazard Safety Level 2guidelines published by the NIH.

The uptake of chimeric GAA is analyzed in (1) COS-7 cells, (2) HL-1cells, and (3) Forbes-Cori and/or Andersen Disease patient cells asdescribed in Example 2 above. COS-7 cells, HL-1 cells, or myocytesand/or hepatocytes from a Forbes-Cori and/or Andersen Disease patientare grown in medium containing 10% FBS and incubated for 40 hours withthe medium of transfected 293 cells producing chimeric hGAA withactivity of 300 nmol/hr/ml. GAA activity and glycogen in culturedpatient myocytes and/or hepatocytes is analyzed as described above.

iii) Immunoblot Detection of Transfected Human GAA, and Assay of GAAMediated Hydrolysis of Glycogen.

The same procedures described in Example 2 are utilized.

Create and Validate cDNA Fv3E10 Genetically Conjugated to GAA (e.g., aGAA Polypeptide Comprising the Amino Acid Sequence of SEQ ID NO: 22)

i) Synthesis of the cDNA for Fv3E10

The cDNA encoding the mouse Fv3E10 variable light chain linked to the3E10 heavy chain (SEQ ID NOs: 9-10) contains a mutation in the VH CDR1that enhances the cell penetrating capacity of the Fv fragment (Zack etal., 1996, J Immunol, 157(5): 2082-8). The 3E10 cDNA is flanked byrestriction sites that facilitate cloning in frame with the cDNA codingsequence that corresponds to the amino acid sequences of the GAApolypeptide (e.g., a GAA polypeptide comprising the amino acid sequenceof SEQ ID NO: 22). The constructs are synthesized and sequenced byGenscript or other qualified manufacturer of gene sequences. To maximizeexpression the 3E10 cDNA is codon optimized for mammalian and pichiaexpression. In the event that mammals or pichia prefer a different codonfor a given amino acid, the next best candidate to unify the preferenceis used. The resulting cDNA is cloned into a mammalian expressioncassette and large scale preps of the plasmid pCMV-3E10-GAA are madeusing the Qiagen Mega Endo-free plasmid purification kit. The constructsare tested in 1) COS-7 cells, 2) HL-1 cells, 3) myofibers and/orhepatocytes from wildtype humans and/or curly coated retrievers, and 4)myofibers and/or hepatocytes from Forbes-Cori Disease humans and/orcurly coated retrievers. The constructs are also tested in a parallelexperiment in 1) COS-7 cells, 2) HL-1 cells, 3) myofibers and/orhepatocytes from wildtype humans, mice and/or Norwegian Forest cats, and4) myofibers and/or hepatocytes from Andersen Disease humans, miceand/or Norwegian Forest cats.

ii) Transfection of Cells

The strategy to test the expression and glycogen hydrolysis of the3E10-GS3- GAA genetic fusion is described above. The transfectionprocedure is the same as described above for transfection of the humanGAA cDNA. Transfected cells are assayed for expression of hGAA andhydrolysis of glycogen as described above.

Production of Recombinant 3E10 Genetically Conjugated to GAA

i) Construction of protein expression vectors for pichia. Plasmidconstruction, transfection, colony selection and culture of Pichia usekits and manuals per the manufacturer's instructions (Invitrogen). ThecDNAs for genetically conjugated 3E10-GS3-GAA created and validated asdescribed above are cloned into two alternative plasmids; PICZ forintracellular expression and PICZalpha for secreted expression. Proteinexpression from each plasmid is driven by the AOX1 promoter. Transfectedpichia is selected with Zeocin and colonies are tested for expression ofrecombinant 3E10-GS3-GAA. High expressers are selected and scaled forpurification.

ii) Purification of Recombinant 3E10-GS3-GAA

cDNA fusions with mAb 3E10 Fv are ligated into the yeast expressionvector pPICZA which is subsequently electroporated into the Pichiapastoris X-33 strain. Colonies are selected with Zeocin (Invitrogen,Carlsbad, Calif.) and identified with anti-his6 antibodies (Qiagen Inc,Valencia, Calif.). X-33 cells are grown in baffled shaker flasks withbuffered glycerol/methanol medium, and protein synthesis is induced with0.5% methanol according to the manufacturer's protocol (EasySelectPichia Expression Kit, Invitrogen, Carlsbad, Calif.). The cells arelysed by two passages through a French Cell Press at 20,000 lbs/in2, andrecombinant protein is purified from cell pellets solubilized in 9Mguanidine HCl and 2% NP40 by immobilized metal ion affinitychromatography (IMAC) on Ni-NTAAgarose (Qiagen, Valencia, Calif.). Boundprotein is eluted in 50 mM NaH2PO4 containing 300 mM NaCl, 500 mMimidazole, and 25% glycerol. Samples of eluted fractions areelectrophoresed in 4-20% gradient SDSPAGE (NuSep Ltd, Frenchs Forest,Australia), and recombinant proteins is identified by Western blottingto nitrocellulose membranes developed with cargo-specific mouseantibodies followed by alkalinephosphatase-conjugated goat antibodies tomouse IgG. Alkaline phosphatase activity is measured by the chromogenicsubstrate, nitroblue tetrazoliumchloride/5-bromo-4-chloro-3-indolylphosphate p-toluidine salt. Proteinsare identified in SDS-PAGE gels with GelCode Blue Stain Reagent (PierceChemical Co., Rockford, Il..). Eluted protein is concentrated,reconstituted with fetal calf serum to 5%, and exchange dialyzed100-fold in 30,000 MWCO spin filters (Millipore Corp., Billerica, Mass.)against McCoy's medium (Mediatech, Inc., Herndon, Va.) containing 5%glycerol. Although in this example a Pichia expression system isillustrated, protein may also be produced in other expression systems,including mammalian expressions systems such as CHO cells. Vectors andmethodologies, including contract manufacturing services, for expressingproteins in CHO cells are available, for example, from Lonza.

iii) Quality Assessment and Formulation

Immunoblot against 3E10 and GAA is used to verify the size and identityof recombinant proteins, followed by silver staining to identify therelative purity among preparations of 3E10, GAA and 3E10-GS3-GAA.Recombinant material is formulated in a buffer and concentration (˜0.5mg/ml).

iv) In Vitro Assessment of Recombinant Material

The activity of 3E10-GS3-GAA protein is evaluated using any one or moreof the assays detailed in Example 2. Cell penetration and/or enzymaticactivity is compared to suitable controls. Moreover, the amount of3E10-GS3-GAA protein needed to alleviate the GAA deficiency isdetermined using the methods described above. The amounts of GAAactivity in mammalian cell-derived and pichia-derived recombinant3E10-GS3-GAA can be tested, for example, on (1) hepatocytes and/ormyocytes from Forbes-Cori and/or Andersen Disease patients and controlpatients, (2) hepatocytes and/or myocytes isolated from wildtype andForbes-Cori Disease curly coated retrievers, (3) hepatocytes and/ormyocytes isolated from wildtype and Andersen Disease Norwegian Forestcats, (4) neutrophils, fibroblasts and/or hepatocytes from wildtypeand/or GSD-Ia and/or GSD-Ib mice and/or von Gierke Disease patientsand/or control patients; and/or (5) fibroblasts from wildtype and LaforaDisease mice and/or from Lafora Disease patients and control patients.

Example 5 In Vivo Assessment of Muscle Targeted GAA in Forbes-CoriDisease Curly-Coated Retrievers Selection of a Forbes-Cori Disease1 DogModel for Evaluation

The Forbes-Cori Disease Curly-Coated Retriever recapitulates humanForbes-Cori Disease in many ways (Yi et al. 2012). These dogs do notmake functional AGL protein (Yi et al., 2012). To control whether asuperphysiological level of GAA is a beneficial treatment, 3E10-GAA isadministered to Forbes-Cori Disease dogs. Selection of dose of GAA

The evaluation dose of 3E10 (e.g., full-length mAb 3E10, Fab-3E10 orFv-3E10) chemically or genetically conjugated to GAA delivered to theForbes-Cori dogs is determined empirically. To minimize the confoundingeffect of a neutralizing immune response to 3E10-GS3-GAA and to maximizethe ability to demonstrate a therapeutic effect, two high doses of 5mg/kg of 3E10-GS3-GAA delivered in one week, followed by assessment ofchanges in disease endpoints, are assessed. The development ofanti-3E10-GAA antibodies is also monitored. Following establishment thatintravenous 3E10*GAA or 3E10-GS3-GAA results in an improvement inglycogen branching defects or aberrant glycogen storage, subsequent invivo assessments in other models (e.g., primates) are initiated,followed by assessment of changes in glycogen debranching defects, asdetermined by immunohistochemistry (e.g., PAS staining).

Materials and Methods

i) Injection of Chemically and Genetically Conjugated 3E10-GAA

3E10*GAA or 3E10-GS3-GAA is formulated and diluted in a buffer that isconsistent with intravenous injection (e.g. sterile saline solution or abuffered solution of 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl). The amount of3E10*GAA or 3E10-GS3-GAA given to each dog is calculated as follows:dose (mg/kg)×dog weight (kg)×stock concentration (mg/ml)=volume (ml) ofstock per dog, q.s. to 100 ul with vehicle.

ii) Blood Collection

Blood is collected by cardiac puncture at the time that animals aresacrificed for tissue dissection. Serum is removed and frozen at -80oC.To minimize the effects of thawing and handling all analysis of 3E10*GAAor 3E10-GS3-GAA circulating in the blood is performed on the same day.

iii) Tissue Collection and Preparation

Sampled tissues are divided for immunoblot, glycogen analysis,formalin-fixed paraffin-embedded tissue blocks and frozen sections inOCT. Heart, liver, lung, spleen, kidneys, quadriceps, EDL, soleus,diaphragm, and biceps tissue (50-100 mg) are subdivided and frozen inplastic tubes for further processing for immunoblot and glycogenanalysis. Additional samples of heart, liver, lung, spleen, kidneys,quadriceps, EDL, soleus, diaphragm, and biceps are subdivided, frozen inOCT tissue sectioning medium, or fixed in 3% glutaraldehyde formaldehydefixation for 24 to 48 hours at 4° C. and embedded in Epon resin, orfixed in 10% NBF and processed into paraffin blocks.

iv) Histological Evaluation

Epon-resin embedded samples are cut at 1μm and stained withPAS-Richardson's stain for glycogen staining. Reduced levels of glycogenaccumulation in tissues (e.g., muscle or liver) of Forbes-Cori dogstreated with 3E10*GAA or 3E10-GS3-GAA as compared to control treatedForbes-Cori dogs is indicative that the 3E10*GAA or 3E10-GS3-GAA iscapable of reducing glycogen levels in vivo.

The paraffin-embedded samples are cut at 1 μm and stained with H&E ortrichrome stains. Reduced fibrosis in liver samples or reduced frayingof myofibrils in muscle samples from Forbes-Cori dogs treated with3E10*GAA or 3E10-GS3-GAA as compared to control treated Forbes-Cori dogsis indicative that the 3E10*GAA or 3E10-GS3-GAA is capable of reducing aliver and/or muscular defect in these dogs.

v) Immunofluorescence

Exogenously delivered GAA is detected using a polyclonal or monoclonalanti-GAA antibody, such as the antibody used in Chen et al., Am J HumGenet. 1987 December; 41(6):1002-15 or Parker et al. (2007).AMP-activated protein kinase does not associate with glycogenalpha-particles from rat liver. Biochem. Biophys. Res. Commun.362:811-815. Ten micrometer frozen sections are cut and placed onSuperfrost Plus microscope slides.

vi) Immunoblot

Immunoblot is used to detect 3E10 and GAA immune reactive material in3E10-GAA treated muscles and hepatic tissues. Protein isolation andimmunoblot detection of 3E10 and GAA is performed according to routineimmunoblot methods. GAA is detected with an antibody specific for thisprotein. Antibody detection of blotted proteins uses NBT/BCIP as asubstrate. Controls include vehicle and treated Forbes-Cori dogs andvehicle and treated homozygous wildtype dogs.

vii) Analysis of Circulating 3E10-GAA

An ELISA specific to human 3E10-GAA is developed and validated usingavailable anti-human GAA antibodies and horseradish peroxidaseconjugated anti-mouse secondary antibody (Jackson Immunoresearch).Recombinant 3E10-GAA is diluted and used to generate a standard curve.Levels of 3E10-GAA are determined from dilutions of serum (normalized tong/ml of serum) or tissue extracts (normalized to ng/mg of tissue).Controls include vehicle and treated wildtype and Forbes-Cori dogs.

viii) Monitoring of Rnti-3E10-GAA Antibody Responses

Purified 3E10-GAA used to inject Forbes-Cori dogs is plated ontohigh-binding 96 well ELISA plates at 1 ug/ml in coating buffer (PierceBiotech), allowed to coat overnight, blocked for 30 minutes in 1% nonfatdrymilk (Biorad) in TBS, and rinsed three times in TBS. Two-folddilutions of sera from vehicle and 3E10-GAA injected animals are loadedinto wells, allowed to incubate for 30 minutes at 37° C., washed threetimes, incubated with horseradish peroxidase (HRP)-conjugated rabbitanti-dog IgA, IgG, and IgM, allowed to incubate for 30 minutes at 37°C., and washed three times. Dog anti-3E10-GAA antibodies are detectedwith TMB liquid substrate and read at 405 nm in ELISA plate reader. Apolyclonal rabbit anti-dog GAA antibody, followed by HRP-conjugated goatanti-rabbit serve as the positive control antibody reaction. Anyabsorbance at 405 nm greater than that of vehicle treated Forbes-Coridogs constitutes a positive anti-3E10-GAA antibody response. Controlsinclude vehicle and treated wildtype dogs and Forbes-Cori dogs.

ix) Assessing Serum Enzyme Levels

Blood is collected from saphenous or jugular veins for each dog everyone to three weeks for the duration of the study. Samples are tested forlevels of alanine transaminase, aspartate transaminase, alkalinephosphatase, and/or creatine phosphokinase. Decrease in the elevatedlevels of one or more of these enzymes is indicative of reduction ofsome of the pathological effects of cytoplasmic glycogen accumulation.

x) Tissue Glycogen Analysis

Tissue glycogen content is assayed enzymatically using the protocoldescribed in Yi et al. (2012). Frozen liver or muscle tissues (50-100mg) are homogenized in ice-cold de-ionized water (20 ml water/g tissue)and sonicated three times for 20 seconds with 30-second intervalsbetween pulses using an ultrasonicator. Homogenates are clarified bycentrifugation at 12,000 g for 20 minutes at 4° C. Supernatant (20 ul)is mixed with 55u1 of water, boiled for 3 minutes and cooled to roomtemperature. Amyloglucosidase (Sigma) solution (25 ul diluted 1:50 into0.1M potassium acetate buffer, pH 5.5) is added and the reactionincubated at 37° C. for 90 minutes. Samples are boiled for 3 minutes tostop the reaction and centrifuged at top speed for 3 minutes in abench-top microcentrifuge. Supernatant (30 ul) is mixed with lml ofInfinity Glucose reagent (Thermo Scientific) and left at roomtemperature for at least 10 minutes. Absorbance at 340nm is measuredusing a UV-1700 spectrophotometer. A reaction without amyloglucosidaseis used for background correction for each sample. A standard curve isgenerated using standard glucose solutions in the reaction with InfinityGlucose reagent (0-120 uM final glucose concentration in the reaction).

xi) Survival Assessment

Those treated and untreated diseased and control dogs that are notsacrificed in the experiments described above are monitored in asurvival study. Specifically, the disease state, treatment conditionsand date of death of the animals are recorded. A survival curve isprepared based on the results of this study.

xii) Statistical Analysis

Pairwise comparisons employ Student's t-test. Comparisons among multiplegroups employ ANOVA. In both cases a p-value<0.05 is consideredstatistically significant.

The foregoing experimental scheme is similarly used to evaluate otherchimeric polypeptides. By way of non-limiting example, this scheme isused to evaluate chemical conjugates and fusion proteins having a GAAportion (or a fragment thereof) and an internalizing moiety portion.

Example 6 In Vivo Assessment of Muscle Targeted GAA in Andersen DiseaseMice Selection of an Andersen Disease Mouse Model for Evaluation

Juvenile and adult onset models of Andersen Disease have been developed.For example, a juvenile and adult onset mouse model of Andersen Diseasewas generated that contains a kinase-neomycin cassette within intron 7of the GBE gene, resulting in decreased GBE expression. This juvenileand adult onset mouse model displays progressive neuromusculardysfunction, aberrant glycogen accumulation in muscle cells andhepatocytes, and shortened lifespan (Akman, et al., 2011). Selection ofdose of GAA

The evaluation dose of 3E10 (e.g., full-length mAb 3E10, Fab-3E10 orFv-3E10) chemically or genetically conjugated to GAA delivered to theAndersen Disease mice is determined empirically. To minimize theconfounding effect of a neutralizing immune response to 3E10-GS3-GAA andto maximize the ability to demonstrate a therapeutic effect, two highdoses of 5 mg/kg of 3E10-GS3-GAA delivered in one week, followed byassessment of changes in disease endpoints, is assessed. The developmentof anti-3E10-GAA antibodies is also be monitored. Followingestablishment that intravenous 3E10*GAA or 3E10-GS3-GAA results in animprovement in aberrant glycogen storage, subsequent in vivo assessmentsin other models (e.g., primates) are initiated, followed by assessmentof changes in glycogen clearance, as determined by immunohistochemistry(e.g., PAS staining). A positive evaluation of 3E10*GAA or 3E10-GS3-GAAwill justify the production of quantities of GLP-grade material neededto perform a more thorough pharmacology and toxicology assessment, andthus determine a dose and dosing range for pre-IND studies.

Materials and Methods

i) Injection of Chemically and Genetically Conjugated 3E10-GAA

3E10*GAA or 3E10-GS3-GAA is formulated and diluted in a buffer that isconsistent with intravenous injection (e.g. sterile saline solution or abuffered solution of 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl). The amount of3E10*GAA or 3E10-GS3-GAA given to each mouse is calculated as follows:dose (mg/kg) x mouse weight (kg) x stock concentration (mg/ml)=volume(ml) of stock per mouse, q.s. to 100 ul with vehicle.

ii) Blood Collection

Blood is collected by cardiac puncture at the time that animals aresacrificed for tissue dissection. Serum is removed and frozen at −80° C.To minimize the effects of thawing and handling all analysis of 3E10*GAAor 3E10-GS3-GAA circulating in the blood is performed on the same day.

iii) Tissue Collection and Preparation

Sampled tissues are divided for immunoblot, glycogen analysis,formalin-fixed paraffin-embedded tissue blocks and frozen sections inOCT. Heart, liver, lung, spleen, kidneys, quadriceps, EDL, soleus,diaphragm, and biceps tissue (50-100 mg) are subdivided and frozen inplastic tubes for further processing for immunoblot and glycogenanalysis. Additional samples of heart, liver, lung, spleen, kidneys,quadriceps, EDL, soleus, diaphragm, and biceps are subdivided, frozen inOCT tissue sectioning medium, or fixed in 3% glutaraldehyde formaldehydefixation for 24 to 48 hours at 4° C. and embedded in Epon resin, orfixed in 10% NBF and processed into paraffin blocks.

iv) Histological Evaluation

Epon-resin embedded samples are cut at 1μm and stained withPAS-Richardson's stain for glycogen staining. Reduced levels of glycogenaccumulation in tissues (e.g., muscle or liver) of Andersen Disease micetreated with 3E10*GAA or 3E10-GS3-GAA as compared to control treatedAndersen Disease mice is indicative that the 3E10*GAA or 3E10-GS3-GAA iscapable of reducing glycogen levels in vivo.

v) Immunofluorescence

Exogenously delivered GAA is detected using a polyclonal or monoclonalanti-GAA antibody, such as the antibody used in Chen et al., Am J HumGenet. 1987 December; 41(6):1002-15 or Parker et al. (2007).AMP-activated protein kinase does not associate with glycogenalpha-particles from rat liver. Biochem. Biophys. Res. Commun.362:811-815. Ten micrometer frozen sections are cut and placed onSuperfrost Plus microscope slides.

vi) Immunoblot

Immunoblot is used to detect 3E10 and GAA immune reactive material in3E10-GAA treated muscles and hepatic tissues. Protein isolation andimmunoblot detection of 3E10 and GAA is performed according to routineimmunoblot methods. GAA is detected with an antibody specific for thisprotein. Antibody detection of blotted proteins use NBT/BCIP as asubstrate. Controls include vehicle and treated Andersen Disease miceand vehicle and treated homozygous wildtype mice.

vii) Analysis of Circulating 3E10-GAA

An ELISA specific to human 3E10-GAA is developed and validated usingavailable anti-human GAA antibodies and horseradish peroxidaseconjugated anti-mouse secondary antibody (Jackson Immunoresearch).Recombinant 3E10-GAA is diluted and used to generate a standard curve.Levels of 3E10-GAA are determined from dilutions of serum (normalized tong/ml of serum) or tissue extracts (normalized to ng/mg of tissue).Controls include vehicle and treated wildtype and Andersen Disease mice.

viii) Monitoring of Anti-3E10-GAA Antibody Responses

Purified 3E10-GAA used to inject Andersen Disease mice is plated ontohigh-binding 96 well ELISA plates at 1 ug/ml in coating buffer (PierceBiotech), allowed to coat overnight, blocked for 30 minutes in 1% nonfatdrymilk (Biorad) in TBS, and rinsed three times in TBS. Two-folddilutions of sera from vehicle and 3E10-GAA injected animals are loadedinto wells, allowed to incubate for 30 minutes at 37° C., washed threetimes, incubated with horseradish peroxidase (HRP)-conjugated rabbitanti-mouse IgA, IgG, and IgM, allowed to incubate for 30 minutes at 37°C., and washed three times. Mouse anti-3E10-GAA antibodies are detectedwith TMB liquid substrate and read at 405 nm in ELISA plate reader. Apolyclonal rabbit anti-mouse GAA antibody, followed by HRP-conjugatedgoat anti-rabbit serve as the positive control antibody reaction. Anyabsorbance at 405 nm greater than that of vehicle treated AndersenDisease mice constitutes a positive anti-3E10-GAA antibody response.Controls include vehicle and treated wildtype mice and Andersen Diseasemice.

ix) Tissue Glycogen Analysis

Tissue glycogen content is assayed using the protocol described in Akman(2011). Samples of frozen muscle and liver tissue (-30-60 mg) are boiledin 200 μl of 30% (wt/vol) KOH for 30 min with occasional shaking. Aftercooling, 67 μl of 0.25 m Na2SO4 and 535 μl of ethanol is added. Next,samples are centrifuged at 14500 g for 20 min at 4° C. to collectglycogen. The glycogen pellet is suspended in water (100 μl), 200 μl ofethanol is added and centrifugation as described above is used toharvest glycogen. This ethanol precipitation step is repeated, and theglycogen pellet is dried in a Speed-Vac. Dried glycogen pellets aresuspended in 100 μl of amyloglucosidase [0.3 mg/ml in 0.2 m sodiumacetate (pH 4.8)] and incubated at 37° C. for 3 h to digest glycogen. Todetermine the glucose concentration in the samples, an aliquot (5 μl) ofdigested glycogen is added to 95 μl of a solution containing 0.3 mtriethanolamine (pH 7.6), 0.4 mm MgCl2, 0.9 mm NADP, 1 mm ATP and 0.1 μgof glucose-6-phosphate dehydrogenase/ml. The absorbance at 340 nm isread before and after the addition of 0.1 μg of hexokinase.

xi) Survival Assessment

Those treated and untreated diseased and control mice that are notsacrificed in the experiments described above are monitored in asurvival study. Specifically, the disease state, treatment conditionsand date of death of the animals is recorded. A survival curve isprepared based on the results of this study.

xii) Statistical Analysis

Pairwise comparisons employ Student's t-test. Comparisons among multiplegroups employ ANOVA. In both cases a p-value <0.05 is consideredstatistically significant.

Example 7 In Vivo Assessment of Muscle Targeted GAA in von GierkeDisease Mice Selection of a von Gierke Disease Mouse Model forEvaluation

Mice engineered to be deficient in G6Pase-α were found to mimic humancases of GSD-Ia (Kim et al., 2007, FEBS Lett., 581(20):3833-38).Specifically, these mice manifest metabolic abnormalities characteristicof disturbed glucose homeostasis and also display markedly increasedlevels of granulocyte colony stimulating factor (G-CSF) andcytokine-induced neutrophil chemoattractant (KC). Any of the chimericpolypeptides disclosed herein can also be tested in any of the otherknown animal models of von Gierke Disease. For example, any of thechimeric polypeptides described herein can alternatively be tested inmouse models similar to those described in Lei et al., 1996, Nat Genet.,13:203-209; Chen et al., 2003, Hum Mol Genet, 12:2547-2558.

Selection of Dose of GAA

The evaluation dose of 3E10 (e.g., full-length mAb 3E10, Fab-3E10 orFv-3E10) chemically or genetically conjugated to GAA delivered to theGSD-Ia mice is determined empirically. To minimize the confoundingeffect of a neutralizing immune response to 3E10-GS3-GAA and to maximizethe ability to demonstrate a therapeutic effect, two high doses of 5mg/kg of 3E10-GS3-GAA delivered in one week, followed by assessment ofchanges in disease endpoints, is assessed. The development ofanti-3E10-GAA antibodies is also monitored. Following establishment thatintravenous 3E10*GAA or 3E10-GS3-GAA results in an improvement inaberrant glycogen storage in mice kidney or liver, and/or improvement inneutropenia, subsequent in vivo assessments in other models (e.g.,primates) are initiated, followed by assessment of changes in glycogenclearance, as determined by immunohistochemistry (e.g., PAS staining).

Materials and Methods

i) Injection of Chemically and Genetically Conjugated 3E10-GAA

3E10*GAA or 3E10-GS3-GAA are formulated and diluted in a buffer that isconsistent with intravenous injection (e.g. sterile saline solution or abuffered solution of 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl). The amount of3E10*GAA or 3E10-GS3-GAA given to each mouse is calculated as follows:dose (mg/kg)×mouse weight (kg)×stock concentration (mg/ml)=volume(ml)ofstock per mouse, q.s. to 100 ul with vehicle.

ii) Blood Collection and Analysis

Blood samples are collected from the tail vein of mice usingEDTA-containing CAPIJECT tubes (TerumoMedical Co., Elkton, Md.) in amanner similar to that described in Kim et al., 2007, FEBS Lett,581(20):3833-3838. Manual 200-cell leukocyte differential counts ofperipheral blood cells are performed on Hema 3 (Fisher Scientific,Pittsburgh, PA.) stained smears as described previously. The cytokines,granulocyte colony stimulating factor (G-CSF) and cytokine-inducedneutrophil chemoattractant (KC) are quantified using Quantikine ELISAkits (R&D Systems Inc., Minneapolis, Minn.). If G-CSF and/or KC levelsare reduced in the blood samples from GSD-Ia mice treated with 3E10*GAAor 3E10-GS3-GAA, then that is indicative that the treatment is effectivein reducing levels of these cytokines in the blood of the GSD-Ia mice.In addition, neutrophil count is also assessed in the blood samples. Kimet al., 2007. If neutrophil cell counts are reduced in three week orolder mice treated with 3E10*GAA or 3E10-GS3-GAA as compared toage-matched untreated control mice, then this is indicative that thetreatment is effective in reducing neutrophilia in GSD-Ia mice.

iii) Tissue Collection and Preparation

Sampled tissues are divided for immunoblot, glycogen analysis,formalin-fixed paraffin-embedded tissue blocks and frozen sections inOCT. Heart, liver, lung, spleen, kidneys, quadriceps, EDL, soleus,diaphragm, and biceps tissue (50-100 mg) are subdivided and frozen inplastic tubes for further processing for immunoblot and glycogenanalysis. Additional samples of heart, liver, lung, spleen, kidneys,quadriceps, EDL, soleus, diaphragm, and biceps are subdivided, frozen inOCT tissue sectioning medium, or fixed in 3% glutaraldehyde formaldehydefixation for 24 to 48 hours at 4 ° C. and embedded in Epon resin, orfixed in 10% NBF and processed into paraffin blocks. For hematoxylin andeosin (H&E) staining, tissues are preserved in 10% neutral bufferedformalin, embedded in paraffin, and sectioned at 4-6 micron thickness.Kim et al., 2007, FEBS Lett, 581(20):3833-3838.

iv) Histological Evaluation

Epon-resin embedded samples are cut at 1 μm and stained withPAS-Richardson's stain for glycogen staining. Reduced levels of glycogenaccumulation in tissues (e.g., muscle or liver) of GSD-Ia Disease micetreated with 3E10*GAA or 3E10-GS3-GAA as compared to control treatedGSD-Ia Disease mice is indicative that the 3E10*GAA or 3E10-GS3-GAA iscapable of reducing glycogen levels in vivo.

v) Immunofluorescence

Exogenously delivered GAA is detected using a polyclonal or monoclonalanti-GAA antibody, such as the antibody used in Chen et al., Am J HumGenet. 1987 December; 41(6):1002-15 or Parker et al. (2007). Tenmicrometer frozen sections are cut and placed on Superfrost Plusmicroscope slides.

vi) Immunoblot

Immunoblot are used to detect 3E10 and GAA immune reactive material in3E10-GAA treated muscles and hepatic tissues. Protein isolation andimmunoblot detection of 3E10 and GAA are performed according to routineimmunoblot methods. GAA are detected with an antibody specific for thisprotein. Antibody detection of blotted proteins use NBT/BCIP as asubstrate. Controls include vehicle and treated GSD-Ia Disease mice andvehicle and treated homozygous wildtype mice.

vii) Analysis of Circulating 3E10-GAA

An ELISA specific to human 3E10-GAA are developed and validated usingavailable anti-human GAA antibodies and horseradish peroxidaseconjugated anti-mouse secondary antibody (Jackson Immunoresearch).Recombinant 3E10-GAA are diluted and used to generate a standard curve.Levels of 3E10-GAA are determined from dilutions of serum (normalized tong/ml of serum) or tissue extracts (normalized to ng/mg of tissue).Controls include vehicle and treated wildtype and GSD-Ia mice.

viii) Monitoring of Anti-3E10-GAA Antibody Responses

Purified 3E10-GAA used to inject GSD-Ia mice are plated ontohigh-binding 96 well ELISA plates at 1 ug/ml in coating buffer (PierceBiotech), allowed to coat overnight, blocked for 30 minutes in 1% nonfatdrymilk (Biorad) in TBS, and rinsed three times in TBS. Two-folddilutions of sera from vehicle and 3E10-GAA injected animals are loadedinto wells, allowed to incubate for 30 minutes at 37° C., washed threetimes, incubated with horseradish peroxidase (HRP)-conjugated rabbitanti-mouse IgA, IgG, and IgM, allowed to incubate for 30 minutes at 37°C., and washed three times. Mouse anti-3E10-GAA antibodies are detectedwith TMB liquid substrate and read at 405 nm in ELISA plate reader. Apolyclonal rabbit anti-mouse GAA antibody, followed by HRP-conjugatedgoat anti-rabbit serve as the positive control antibody reaction. Anyabsorbance at 405 nm greater than that of vehicle treated GSD-Ia miceconstitutes a positive anti-3E10-GAA antibody response. Controls includevehicle and treated wildtype mice and GSD-Ia mice.

ix) Tissue Glycogen Analysis

Tissue glycogen content is assayed using the protocol described in Akman(2011). Samples of frozen muscle and liver tissue (˜30-60 mg) are boiledin 200 μl of 30% (wt/vol) KOH for 30 min with occasional shaking. Aftercooling, 67 μl of 0.25 m Na2SO4 and 535 μl of ethanol is added. Next,samples are centrifuged at 14500 g for 20 min at 4° C. to collectglycogen. The glycogen pellet is suspended in water (100 μl), 200 μl ofethanol is added and centrifugation as described above is used toharvest glycogen. This ethanol precipitation step is repeated, and theglycogen pellet is dried in a Speed-Vac. Dried glycogen pellets issuspended in 100 μl of amyloglucosidase [0.3 mg/ml in 0.2 m sodiumacetate (pH 4.8)] and incubated at 37° C. for 3 h to digest glycogen. Todetermine the glucose concentration in the samples, an aliquot (5 μl) ofdigested glycogen is added to 95 μl of a solution containing 0.3 mtriethanolamine (pH 7.6), 0.4 mm MgCl2, 0.9 mm NADP, 1 mm ATP and 0.1 μgof glucose-6-phosphate dehydrogenase/ml. The absorbance at 340 nm isread before and after the addition of 0.1 μg of hexokinase.

xii) Statistical Analysis

Pairwise comparisons employ Student's t-test. Comparisons among multiplegroups employs ANOVA. In both cases a p-value<0.05 is consideredstatistically significant.

Example 8 In Vivo Assessment of Muscle Targeted GAA in Lafora DiseaseMice Selection of a Lafora Disease Mouse Model for Evaluation

Mice engineered to be deficient in malin display a phenotype similar tothat observed in human cases of Lafora Disease. Specifically,matin^(−/−) mice presented in an age-dependent manner neurodegeneration,increased synaptic excitability, and propensity to suffer myoclonicseizures. Valles-Ortega et al., 2011, EMBO Mol Med, 3(11):667-681. Inaddition, these mice accumulated glycogen-filled inclusion bodies thatwere most abundant in the hippocampus and cerebellum, but that were alsofound in skeletal and cardiac muscle cells. Valles-Ortega et al.Glycogen was also found to be less branched in the cells of malin^(−/−)mice as compared to glycogen observed in the cells of healthy controlmice. Valles-Ortega et al. An increased level of glycogenhyperphosphorylation has also been described in this mouse model.Turnbull et al., 2010, Ann Neurol, 68(6):925-33. Alternative mousemodels that could be used in the in vivo experiments described hereininclude the laforin^(−/−) mouse model described in Ganesh et al., 2002,Hum Mol Genet, 11(11):1251-62.

Selection of Dose of GAA

The evaluation dose of 3E10 (e.g., full-length mAb 3E10, Fab-3E10 orFv-3E10) chemically or genetically conjugated to GAA delivered to theGSD-Ia mice is determined empirically. To minimize the confoundingeffect of a neutralizing immune response to 3E10-GS3-GAA and to maximizethe ability to demonstrate a therapeutic effect, two high doses of 5mg/kg of 3E10-GS3-GAA are delivered in one week, followed by assessmentof changes in disease endpoints, are assessed. The development ofanti-3E10-GAA antibodies are also monitored. Following establishmentthat intravenous 3E10*GAA or 3E10-GS3-GAA results in an improvement inaberrant glycogen storage in mice kidney or liver, subsequent in vivoassessments in other models (e.g., primates) are initiated, followed byassessment of changes in glycogen clearance, as determined byimmunohistochemistry (e.g., PAS staining).

Materials and Methods

i) Injection of Chemically and Genetically Conjugated 3E10-GAA

3E10*GAA or 3E10-GS3-GAA are formulated and diluted in a buffer that isconsistent with intravenous injection (e.g. sterile saline solution or abuffered solution of 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl). The amount of3E10*GAA or 3E10-GS3-GAA given to each mouse are calculated as follows:dose (mg/kg)×mouse weight (kg)×stock concentration (mg/ml)=volume (ml)of stock per mouse, q.s. to 100 ul with vehicle.

ii) Blood Collection

Blood is collected by cardiac puncture at the time that animals aresacrificed for tissue dissection. Serum is removed and frozen at −80° C.To minimize the effects of thawing and handling all analysis of 3E10*GAAor 3E10-GS3-GAA circulating in the blood is performed on the same day.

iii) Tissue Collection and Preparation

Sampled tissues are divided for immunoblot, glycogen analysis,formalin-fixed paraffin-embedded tissue blocks and frozen sections inOCT. Heart, liver, lung, spleen, kidneys, quadriceps, EDL, soleus,diaphragm, and biceps tissue (50-100 mg) are subdivided and frozen inplastic tubes for further processing for immunoblot and glycogenanalysis. Additional samples of heart, liver, lung, spleen, kidneys,quadriceps, EDL, soleus, diaphragm, and biceps are subdivided, frozen inOCT tissue sectioning medium, or fixed in 3% glutaraldehyde formaldehydefixation for 24 to 48 hours at 4° C. and embedded in Epon resin, orfixed in 10% NBF and processed into paraffin blocks. Some samples arehomogenized in 30% KOH for 15 min and glycogen levels are determinedusing an amyloglucosidase-based assay described in Valles-Ortega et al.In addition, glycogen branching are assessed in the homogenized samplesusing the methods described in Valles-Ortega et al. A reduction inglycogen accumulation and an increase in glycogen branching in samplesfrom mice treated with 3E10*GAA or 3E10-GS3-GAA as compared to untreatedcontrol mice is indicative that the chimeric polypeptides are capable ofclearing glycogen and improving glycogen branching in the cells of themice.

iv) Histological Evaluation

Epon-resin embedded samples are cut at 1 μm and stained withPAS-Richardson's stain for glycogen staining. Reduced levels of glycogenaccumulation in tissues (e.g., muscle or liver) of Lafora Disease micetreated with 3E10*GAA or 3E10-GS3-GAA as compared to control treatedLafora Disease mice is indicative that the 3E10*GAA or 3E10-GS3-GAA iscapable of reducing glycogen levels in vivo.

v) Immunofluorescence

Exogenously delivered GAA are detected using a polyclonal or monoclonalanti-GAA antibody, such as the antibody used in Chen et al., Am J HumGenet. 1987 December; 41(6):1002-15 or Parker et al. (2007). Tenmicrometer frozen sections are cut and placed on Superfrost Plusmicroscope slides.

vi) Immunoblot

Immunoblot are used to detect 3E10 and GAA immune reactive material in3E10-GAA treated muscles and hepatic tissues. Protein isolation andimmunoblot detection of 3E10 and GAA are performed according to routineimmunoblot methods. GAA are detected with an antibody specific for thisprotein. Antibody detection of blotted proteins use NBT/BCIP as asubstrate. Controls include vehicle and treated Lafora Disease mice andvehicle and treated homozygous wildtype mice.

vii) Analysis of Circulating 3E10-GAA

An ELISA specific to human 3E10-GAA is developed and validated usingavailable anti-human GAA antibodies and horseradish peroxidaseconjugated anti-mouse secondary antibody (Jackson Immunoresearch).Recombinant 3E10-GAA is diluted and used to generate a standard curve.Levels of 3E10-GAA are determined from dilutions of serum (normalized tong/ml of serum) or tissue extracts (normalized to ng/mg of tissue).Controls include vehicle and treated wildtype and GSD-Ia mice.

viii) Monitoring of Anti-3E10-GAA Antibody Responses

Purified 3E10-GAA used to inject GSD-Ia mice are plated ontohigh-binding 96 well ELISA plates at 1 ug/ml in coating buffer (PierceBiotech), allowed to coat overnight, blocked for 30 minutes in 1% nonfatdrymilk (Biorad) in TBS, and rinsed three times in TBS. Two-folddilutions of sera from vehicle and 3E10-GAA injected animals are loadedinto wells, allowed to incubate for 30 minutes at 37° C., washed threetimes, incubated with horseradish peroxidase (HRP)-conjugated rabbitanti-mouse IgA, IgG, and IgM, allowed to incubate for 30 minutes at 37°C., and washed three times. Mouse anti-3E10-GAA antibodies are detectedwith TMB liquid substrate and read at 405 nm in ELISA plate reader. Apolyclonal rabbit anti-mouse GAA antibody, followed by HRP-conjugatedgoat anti-rabbit serve as the positive control antibody reaction. Anyabsorbance at 405 nm greater than that of vehicle treated Lafora miceconstitutes a positive anti-3E10-GAA antibody response. Controls includevehicle and treated wildtype mice and Lafora mice.

ix) Tissue Glycogen Analysis

Tissue glycogen content is assayed using the protocol described in Akman(2011). Samples of frozen muscle and liver tissue (-30-60 mg) are boiledin 200 μl of 30% (wt/vol) KOH for 30 min with occasional shaking. Aftercooling, 67 μl of 0.25 m Na2SO4 and 535 μl of ethanol is added. Next,samples are centrifuged at 14500g for 20 min at 4° C. to collectglycogen. The glycogen pellet is suspended in water (100 μl), 200 μl ofethanol are added and centrifugation as described above is used toharvest glycogen. This ethanol precipitation step is repeated, and theglycogen pellet is dried in a Speed-Vac. Dried glycogen pellets issuspended in 100 μl of amyloglucosidase [0.3 mg/ml in 0.2 m sodiumacetate (pH 4.8)] and incubated at 37° C. for 3 h to digest glycogen. Todetermine the glucose concentration in the samples, an aliquot (5 μl) ofdigested glycogen is added to 95 μl of a solution containing 0.3 mtriethanolamine (pH 7.6), 0.4 mm MgCl2, 0.9 mm NADP, 1 mm ATP and 0.1 μgof glucose-6-phosphate dehydrogenase/ml. The absorbance at 340 nm isread before and after the addition of 0.1 μg of hexokinase.

x) Seizure Assessment

The malin^(−/−) mice described by Valles-Ortega et al. were generated inthe C57BL6 strain of mice, which are normally resistant to seizures.However, while administration of kainate did not induce any seizures inwildtype C57BL6 mice, malin^(−/−) mice treated with kainate displayedclonic hippocampal seizures. Valles-Ortega et al. Malin^(−/−) mice aretreated with kainate and with or without 3E10*GAA or 3E10-GS3-GAA. Ifthe mice treated with kainate and 3E10*GAA or 3E10-GS3-GAA displayreduced seizures as compared to malin^(−/−) mice treated with kainatebut not with any chimeric polypeptides, this is indicative that thechimeric polypeptides are effective in treating some of the neurologicaldefects observed in the malin^(−/−) mice.

xi) Neurodegeneration Analysis

The total number of parvalbumin positive interneurons is assessed in thehippocampus of malin^(−/−) mice treated with or without 3E10*GAA or3E10-GS3-GAA. Valles-Ortega et al. If the hippocampi from mice treatedwith 3E10*GAA or 3E10-GS3-GAA display less parvalbumin-positiveneurodegeneration than in the hippocampi from untreated mice, than thisis indicative that the chimeric polypeptides are effective in reducingneurodegeneration in the malin^(−/−) mice.

xii) Statistical Analysis

Pairwise comparisons employs Student's t-test. Comparisons amongmultiple groups employ ANOVA. In both cases a p-value<0.05 is consideredstatistically significant.

The foregoing experimental scheme (any one or more than one of theforegoing examples) will similarly be used to evaluate other chimericpolypeptides of the disclosure. By way of non-limiting example, thisscheme is used to evaluate chemical conjugates and recombinantconjugates having a GAA portion (or a fragment thereof) and aninternalizing moiety portion. In certain embodiments, the chimericpolypeptide comprises an internalizing moiety that is an antibody orantigen binding fragment. In certain embodiments, the internalizingmoiety is a Fab or Fab′. Any chimeric polypeptide having any GAAportion, as described herein, and any internalizing moiety portion, asdescribed herein, is similarly made and analyzed.

Similar examples to those described above are performed using any of theother chimeric polypeptides disclosed herein (e.g., a chimericpolypeptide comprising a laforin polypeptide portion, or a chimericpolypeptide comprising a malin polypeptide portion, or a chimericpolypeptide comprising an alpha-amylase polypeptide portion, or achimeric polypeptide comprising an AGL polypeptide portion). Forexample, chimeric polypeptides comprising any the laforin polypeptide,as described herein, and any internalizing moiety, as described herein,are made and analyzed. By way of further example, chimeric polypeptidescomprising any AGL polypeptide, as described herein, and anyinternalizing moiety, as described herein, are made and tested. By wayof further example, chimeric polypeptides comprising any malinpolypeptide, as described herein, and any internalizing moiety, asdescribed herein, are made and tested. By way of further example,chimeric polypeptides comprising any alpha-amylase polypeptide, asdescribed herein, and any internalizing moiety, as described herein, aremade and tested.

Exemplary Sequences SEQ ID NO: 1 = full-length, immature GAA aminoacid sequence (952 amino acids; signal sequenceindicated in bold/underline)MGVRHPPCSHRLLAVCALVSLATAALLGHILLHDFLLVPRELSGSSPVLE ETHPAHQQGASRPGPRDAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVS WCSEQ ID NO: 2 = full-length, immature GAA aminoacid sequence (957 amino acids; signal sequenceindicated in bold/underline) (GenBank Accession No. EAW89583.1)MGVRHPPCSHRLLAVCALVSLATAALLGHILLHDFLLVPRELSGSSPVLE ETHPAHQQGASRPGPRDAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPIEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKARGPRVLDICVSLLMGE QFLVSWCSEQ ID NO: 3 = exemplary mature GAA amino acidsequence (corresponding to residues 123-782 ofSEQ ID NO: 1; one embodiment of a mature GAA polypeptide)GQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTW YDLQTVPVEASEQ ID NO: 4 = exemplary mature GAA amino acidsequence (corresponding to residues 288-782 ofSEQ ID NO: 1; one embodiment of a mature GAA polypeptide)GANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEA SEQ ID NO: 5 = G53 linkerGGGGSGGGGSGGGGS SEQ ID NO: 6 = Linker GSTSGSGKSSEGKGSEQ ID NO: 7 = His tag HHHHHHH SEQ ID NO: 8 = c-Myc tag EQKLISEEDLSEQ ID NO: 9 = exemplary 3E10 Variable HeavyChain (V_(H) having D31N substitution; see examples)EVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRG LLLDYWGQGTTLTVSSSEQ ID NO: 10 = 3E10 Variable Light Chain (V_(L))DIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFHLNIHPVEEEDAATYYCQHSREFPW TFGGGTKLELKSEQ ID NO: 11 = Exemplary chimeric polypeptide, Fv3E10-GAA (123-782)DIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFHLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSEQKLSEEDLGSTSGSGKSSEGKGGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPRVHSRAPSPLYSVEFSEEPFGVIVHRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRLYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPIEAHHHHHHSEQ ID NO: 12 = Exemplary chimeric polypeptide, Fv3E10-GAA (288-782)DIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFHLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSEQKLSEEDLGSTSGSGKSSEGKGGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRLYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTW YDLQTVPIEAHHHHHHSEQ ID NO: 13 -heavy chain variable domain CDR1of 3E10 VH (as that VH is defined with referenceto SEQ ID NO: 9), in accordance with Kabat system NYGMHSEQ ID NO: 14 -heavy chain variable domain CDR2of 3E10 VH (as that VH is defined with referenceto SEQ ID NO: 9), in accordance with Kabat system YISSGSSTIYYADTVKGSEQ ID NO: 15 -heavy chain variable domain CDR3of 3E10 VH (as that VH is defined with referenceto SEQ ID NO: 9), in accordance with Kabat system RGLLLDYSEQ ID NO: 16 - light chain variable domain CDR1of 3E10 VL (as that VL is defined with referenceto SEQ ID NO: 10), in accordance with Kabat system RASKSVSTSSYSYMHSEQ ID NO: 17 - light chain variable domain CDR2of 3E10 VL (as that VL is defined with referenceto SEQ ID NO: 10), in accordance with Kabat system YASYLESSEQ ID NO: 18 - light chain variable domain CDR3of 3E10 VL (as that VL is defined with referenceto SEQ ID NO: 10), in accordance with Kabat system QHSREFPWTSEQ ID NO: 19 AGIH SEQ ID NO: 20 SAGIHSEQ ID NO: 21- Exemplary GAA polypeptidecomprising mature GAA (residues 61-952;one embodiment of a GAA polypeptide)SRPGPRDAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWCSEQ ID NO: 22- Exemplary GAA polypeptidecomprising mature GAA (residues 67-952;one embodiment of a GAA polypeptide)DAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWCSEQ ID NO: 23- Exemplary GAA polypeptidecomprising mature GAA (residues 70-952;one embodiment of a GAA polypeptide)AHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWCSEQ ID NO: 24 -heavy chain variable (VH) domainCDR1 of exemplary 3E10 V_(H) (as that VH is definedwith reference to SEQ ID NO: 9), in accordance with the IMGT systemGFTFSNYG SEQ ID NO: 25 - heavy chain variable (VH) domainCDR2 of exemplary 3E10 V_(H) (as that VH is definedwith reference to SEQ ID NO: 9), in accordance with the IMGT systemISSGSSTI SEQ ID NO: 26 - heavy chain variable (VH) domainCDR3 of exemplary 3E10 V_(H) (as that VH is definedwith reference to SEQ ID NO: 9), in accordance with the IMGT systemARRGLLLDY SEQ ID NO: 27 - light chain variable (VL) domainCDR1 of exemplary 3E10 V_(L) (as that VL is definedwith reference to SEQ ID NO: 10), in accordance with the IMGT systemKSVSTSSYSY SEQ ID NO: 28 - light chain variable (VL) domainCDR2 of exemplary 3E10 V_(L) (as that VL is definedwith reference to SEQ ID NO: 10), in accordance with the IMGT system YASSEQ ID NO: 29 - light chain variable (VL) domainCDR3 of exemplary 3E10 V_(L) (as that VL is definedwith reference to SEQ ID NO: 10), in accordance with the IMGT systemQHSREFPWT SEQ ID NO: 30- linker sequence GGSGGGSGGGSGGSEQ ID NO: 31- full linker region (residues 57-78 of GAA)HILLHDFLLVPRELSGSSPVLEETHPAH SEQ ID NO: 32- bovine GAA precursor protein(GenBank Accession No. NP_776338.1)MMRWPPCSRPLLGVCTLLSLALLGHILLHDLEVVPRELRGFSQDEIHQACQPGASSPECRGSPRAAPTQCDLPPNSRFDCAPDKGITPQQCEARGCCYMPAEWPPDAQMGQPWCFFPPSYPSYRLENLTTTETGYTATLTRAVPTFFPKDIMTLRLDMLMETESRLHFTIKDPANRRYEVPLETPRVYSQAPFTLYSVEFSEEPFGVVVRRKLDGRVLLNTTVAPLFFADQFLQLSTSLPSQHITGLAEHLGSLMLSTNWTKITLWNRDIAPEPNVNLYGSHPFYLVLEDGGLAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSTSAITRQVVENMTRAYFPLDVQWNDLDYMDARRDFTFNKDHFGDFPAMVQELHQGGRRYIMIVDPAISSSGPAGTYRPYDEGLRRGVFITNETGQPLIGQVWPGLTAFPDFTNPETLDWWQDMVTEFHAQVPFDGMWIDMNEPSNFVRGSVDGCPDNSLENPPYLPGVVGGTLRAATICASSHQFLSTHYDLHNLYGLTEALASHRALVKARGMRPFVISRSTFAGHGRYSGHWTGDVWSNWEQLSYSVPEILLFNLLGVPLVGADICGFLGNTSEELCVRWTQLGAFYPFMRNHNALNSQPQEPYRFSETAQQAMRKAFTLRYVLLPYLYTLFHRAHVRGETVARPLFLEFPEDPSTWTVDRQLLWGEALLITPVLEAEKVEVTGYFPQGTWYDLQTVPMEAFGSLPPPAPLTSVIHSKGQWVTLSAPLDTINVHLRAGHIIPMQGPALTTTESRKQHMALAVALTASGEAQGELFWDDGESLGVLDGGDYTQLIFLAKNNTFVNKLVHVSSEGASLQLRNVTVLGVATAPQQVLCNSVPVSNFTFSPDTETLAIPVSLTMGEQFVISWSSEQ ID NO: 33- Exemplary Signal Sequence MSVPTQVLGLLLLWLTDARCSEQ ID NO: 34- murine kappa constant domain (CL)RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVK SFNRNECSEQ ID NO: 35- mu3E10 light chain sequence (VL + CL) + signal sequenceMSVPTQVLGLLLLWLTDARCDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFHLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECUnderlined sequence corresponds to murine signalsequence; Bolded sequence corresponds to murine kappa constant domainSEQ ID NO: 36- Exemplary Signal Sequence MEWSWVFLFFLSVTTGVHSSEQ ID NO: 37- mu3E10 variable heavy chainsequence (VH) + signal sequenceMEWSWVFLFFLSVTTGVHSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSUnderlined sequence corresponds to murine signal sequenceSEQ ID NO: 38- human laforin (EPM2A) isoform a(GenBank Accession No. NM_005670.3)MRFRFGVVVPPAVAGARPELLVVGSRPELGRWEPRGAVRLRPAGTAAGDGALALQEPGLWLGEVELAAEEAAQDGAEPGRVDTFWYKFLKREPGGELSWEGNGPHHDRCCTYNENNLVDGVYCLPIGHWIEATGHTNEMKHTTDFYFNIAGHQAMHYSRILPNIWLGSCPRQVEHVTIKLKHELGITAVMNFQTEWDIVQNSSGCNRYPEPMTPDTMIKLYREEGLAYIWMPTPDMSTEGRVQMLPQAVCLLHALLEKGHIVYVHCNAGVGRSTAAVCGWLQYVMGWNLRKVQYFLMAKRPAVYIDEEALARAQEDFFQKFGKVRSSVCSLSEQ ID NO: 39- human laforin (EPM2A) isoform b(GenBank Accession No. NM_001018041.1)MRFRFGVVVPPAVAGARPELLVVGSRPELGRWEPRGAVRLRPAGTAAGDGALALQEPGLWLGEVELAAEEAAQDGAEPGRVDTFWYKFLKREPGGELSWEGNGPHHDRCCTYNENNLVDGVYCLPIGHWIEATGHTNEMKHTTDFYFNIAGHQAMHYSRILPNIWLGSCPRQVEHVTIKLKHELGITAVMNFQTEWDIVQNSSGCNRYPEPMTPDTMIKLYREEGLAYIWMPTPDMSTEGRVQMLPQAVCLLHALLEKGHIVYVHCNAGVGRSTAAVCGWLQYVMGWNLRKVQYFLMAKR PAVYIDEEAASQDTFPLSEQ ID NO: 40- The amino acid sequence of thehuman AGL protein, isoform 1 (GenBank Accession No. NP_000019.2)MGHSKQIRILLLNEMEKLEKTLFRLEQGYELQFRLGPTLQGKAVTVYTNYPFPGETFNREKFRSLDWENPTEREDDSDKYCKLNLQQSGSFQYYFLQGNEKSGGGYIVVDPILRVGADNHVLPLDCVTLQTFLAKCLGPFDEWESRLRVAKESGYNMIHFTPLQTLGLSRSCYSLANQLELNPDFSRPNRKYTWNDVGQLVEKLKKEWNVICITDVVYNHTAANSKWIQEHPECAYNLVNSPHLKPAWVLDRALWRFSCDVAEGKYKEKGIPALIENDHHMNSIRKIIWEDIFPKLKLWEFFQVDVNKAVEQFRRLLTQENRRVTKSDPNQHLTIIQDPEYRRFGCTVDMNIALTTFIPHDKGPAAIEECCNWFHKRMEELNSEKHRLINYHQEQAVNCLLGNVFYERLAGHGPKLGPVTRKHPLVTRYFTFPFEEIDFSMEESMIHLPNKACFLMAHNGWVMGDDPLRNFAEPGSEVYLRRELICWGDSVKLRYGNKPEDCPYLWAHMKKYTEITATYFQGVRLDNCHSTPLHVAEYMLDAARNLQPNLYVVAELFTGSEDLDNVFVTRLGISSLIREAMSAYNSHEEGRLVYRYGGEPVGSFVQPCLRPLMPAIAHALFMDITHDNECPIVHRSAYDALPSTTIVSMACCASGSTRGYDELVPHQISVVSEERFYTKWNPEALPSNTGEVNFQSGIIAARCAISKLHQELGAKGFIQVYVDQVDEDIVAVTRHSPSIHQSVVAVSRTAFRNPKTSFYSKEVPQMCIPGKIEEVVLEARTIERNTKPYRKDENSINGTPDITVEIREHIQLNESKIVKQAGVATKGPNEYIQEIEFENLSPGSVIIFRVSLDPHAQVAVGILRNHLTQFSPHFKSGSLAVDNADPILKIPFASLASRLTLAELNQILYRCESEEKEDGGGCYDIPNWSALKYAGLQGLMSVLAEIRPKNDLGHPFCNNLRSGDWMIDYVSNRLISRSGTIAEVGKWLQAMFFYLKQIPRYLIPCYFDAILIGAYTTLLDTAWKQMSSFVQNGSTFVKHLSLGSVQLCGVGKFPSLPILSPALMDVPYRLNEITKEKEQCCVSLAAGLPHFSSGIFRCWGRDTFIALRGILLITGRYVEARNIILAFAGTLRHGLIPNLLGEGIYARYNCRDAVWWWLQCIQDYCKMVPNGLDILKCPVSRMYPTDDSAPLPAGTLDQPLFEVIQEAMQKHMQGIQFRERNAGPQIDRNMKDEGFNITAGVDEETGFVYGGNRFNCGTWMDKMGESDRARNRGIPATPRDGSAVEIVGLSKSAVRWLLELSKKNIFPYHEVTVKRHGKAIKVSYDEWNRKIQDNFEKLFHVSEDPSDLNEKHPNLVHKRGIYKDSYGASSPWCDYQLRPNFTIAMVVAPELFTTEKAWKALEIAEKKLLGPLGMKTLDPDDMVYCGIYDNALDNDNYNLAKGFNYHQGPEWLWPIGYFLRAKLYFSRLMGPETTAKTIVLVKNVLSRHYVHLERSPWKGLPELTNENAQYCPFSCETQAWSIATILETLYDLSEQ ID NO: 41- The amino acid sequence of thehuman AGL protein, isoform 2 (GenBank Accession No. NM_000645.2)MSLLTCAFYLGYELQFRLGPTLQGKAVTVYTNYPFPGETFNREKFRSLDWENPTEREDDSDKYCKLNLQQSGSFQYYFLQGNEKSGGGYIVVDPILRVGADNHVLPLDCVTLQTFLAKCLGPFDEWESRLRVAKESGYNMIHFTPLQTLGLSRSCYSLANQLELNPDFSRPNRKYTWNDVGQLVEKLKKEWNVICITDVVYNHTAANSKWIQEHPECAYNLVNSPHLKPAWVLDRALWRFSCDVAEGKYKEKGIPALIENDHHMNSIRKIIWEDIFPKLKLWEFFQVDVNKAVEQFRRLLTQENRRVTKSDPNQHLTIIQDPEYRRFGCTVDMNIALTTFIPHDKGPAAIEECCNWFHKRMEELNSEKHRLINYHQEQAVNCLLGNVFYERLAGHGPKLGPVTRKHPLVTRYFTFPFEEIDFSMEESMIHLPNKACFLMAHNGWVMGDDPLRNFAEPGSEVYLRRELICWGDSVKLRYGNKPEDCPYLWAHMKKYTEITATYFQGVRLDNCHSTPLHVAEYMLDAARNLQPNLYVVAELFTGSEDLDNVFVTRLGISSLIREAMSAYNSHEEGRLVYRYGGEPVGSFVQPCLRPLMPAIAHALFMDITHDNECPIVHRSAYDALPSTTIVSMACCASGSTRGYDELVPHQISVVSEERFYTKWNPEALPSNTGEVNFQSGIIAARCAISKLHQELGAKGFIQVYVDQVDEDIVAVTRHSPSIHQSVVAVSRTAFRNPKTSFYSKEVPQMCIPGKIEEVVLEARTIERNTKPYRKDENSINGTPDITVEIREHIQLNESKIVKQAGVATKGPNEYIQEIEFENLSPGSVIIFRVSLDPHAQVAVGILRNHLTQFSPHFKSGSLAVDNADPILKIPFASLASRLTLAELNQILYRCESEEKEDGGGCYDIPNWSALKYAGLQGLMSVLAEIRPKNDLGHPFCNNLRSGDWMIDYVSNRLISRSGTIAEVGKWLQAMFFYLKQIPRYLIPCYFDAILIGAYTTLLDTAWKQMSSFVQNGSTFVKHLSLGSVQLCGVGKFPSLPILSPALMDVPYRLNEITKEKEQCCVSLAAGLPHFSSGIFRCWGRDTFIALRGILLITGRYVEARNIILAFAGTLRHGLIPNLLGEGIYARYNCRDAVWWWLQCIQDYCKMVPNGLDILKCPVSRMYPTDDSAPLPAGTLDQPLFEVIQEAMQKHMQGIQFRERNAGPQIDRNMKDEGFNITAGVDEETGFVYGGNRFNCGTWMDKMGESDRARNRGIPATPRDGSAVEIVGLSKSAVRWLLELSKKNIFPYHEVTVKRHGKAIKVSYDEWNRKIQDNFEKLFHVSEDPSDLNEKHPNLVHKRGIYKDSYGASSPWCDYQLRPNFTIAMVVAPELFTTEKAWKALEIAEKKLLGPLGMKTLDPDDMVYCGIYDNALDNDNYNLAKGFNYHQGPEWLWPIGYFLRAKLYFSRLMGPETTAKTIVLVKNVLSRHYVHLERSPWKGLPELTNENAQYCPFSCET QAWSIATILETLYDLSEQ ID NO: 42- The amino acid sequence of thehuman AGL protein, isoform 3 (GenBank Accession No. NM_000646.2)MAPILSINLFIGYELQFRLGPTLQGKAVTVYTNYPFPGETFNREKFRSLDWENPTEREDDSDKYCKLNLQQSGSFQYYFLQGNEKSGGGYIVVDPILRVGADNHVLPLDCVTLQTFLAKCLGPFDEWESRLRVAKESGYNMIHFTPLQTLGLSRSCYSLANQLELNPDFSRPNRKYTWNDVGQLVEKLKKEWNVICITDVVYNHTAANSKWIQEHPECAYNLVNSPHLKPAWVLDRALWRFSCDVAEGKYKEKGIPALIENDHHMNSIRKIIWEDIFPKLKLWEFFQVDVNKAVEQFRRLLTQENRRVTKSDPNQHLTIIQDPEYRRFGCTVDMNIALTTFIPHDKGPAAIEECCNWFHKRMEELNSEKHRLINYHQEQAVNCLLGNVFYERLAGHGPKLGPVTRKHPLVTRYFTFPFEEIDFSMEESMIHLPNKACFLMAHNGWVMGDDPLRNFAEPGSEVYLRRELICWGDSVKLRYGNKPEDCPYLWAHMKKYTEITATYFQGVRLDNCHSTPLHVAEYMLDAARNLQPNLYVVAELFTGSEDLDNVFVTRLGISSLIREAMSAYNSHEEGRLVYRYGGEPVGSFVQPCLRPLMPAIAHALFMDITHDNECPIVHRSAYDALPSTTIVSMACCASGSTRGYDELVPHQISVVSEERFYTKWNPEALPSNTGEVNFQSGIIAARCAISKLHQELGAKGFIQVYVDQVDEDIVAVTRHSPSIHQSVVAVSRTAFRNPKTSFYSKEVPQMCIPGKIEEVVLEARTIERNTKPYRKDENSINGTPDITVEIREHIQLNESKIVKQAGVATKGPNEYIQEIEFENLSPGSVIIFRVSLDPHAQVAVGILRNHLTQFSPHFKSGSLAVDNADPILKIPFASLASRLTLAELNQILYRCESEEKEDGGGCYDIPNWSALKYAGLQGLMSVLAEIRPKNDLGHPFCNNLRSGDWMIDYVSNRLISRSGTIAEVGKWLQAMFFYLKQIPRYLIPCYFDAILIGAYTTLLDTAWKQMSSFVQNGSTFVKHLSLGSVQLCGVGKFPSLPILSPALMDVPYRLNEITKEKEQCCVSLAAGLPHFSSGIFRCWGRDTFIALRGILLITGRYVEARNIILAFAGTLRHGLIPNLLGEGIYARYNCRDAVWWWLQCIQDYCKMVPNGLDILKCPVSRMYPTDDSAPLPAGTLDQPLFEVIQEAMQKHMQGIQFRERNAGPQIDRNMKDEGFNITAGVDEETGFVYGGNRFNCGTWMDKMGESDRARNRGIPATPRDGSAVEIVGLSKSAVRWLLELSKKNIFPYHEVTVKRHGKAIKVSYDEWNRKIQDNFEKLFHVSEDPSDLNEKHPNLVHKRGIYKDSYGASSPWCDYQLRPNFTIAMVVAPELFTTEKAWKALEIAEKKLLGPLGMKTLDPDDMVYCGIYDNALDNDNYNLAKGFNYHQGPEWLWPIGYFLRAKLYFSRLMGPETTAKTIVLVKNVLSRHYVHLERSPWKGLPELTNENAQYCPFSCE TQAWSIATILETLYDLSEQ ID NO: 43 - Human malin Amino Acid Sequence(GenBank Accession No. AY324850.1)MAAEASESGPALHELMREAEISLLECKVCFEKFGHRQQRRPRNLSCGHVVCLACVAALAHPRTLALECPFCRRACRGCDTSDCLPVLHLIELLGSALRQSPAAHRAAPSALGALTCHHTFGGWGTLVNPTGLALCPKTGRVVVVHDGRRRVKIFDSGGGCAHQFGEKGDAAQDIRYPVDVTITNDCHVVVTDAGDRSIKVFDFFGQIKLVIGGQFSLPWGVETTPQNGIVVTDAEAGSLHLLDVDFAEGVLRRTERLQAHLCNPRGVAVSWLTGAIAVLEHPLALGTGVCSTRVKVFSSSMQLVGQVDTFGLSLYFPSKITASAVTFDHQGNVIVADTSGPAILCLGKPEEFPVPKPMVTHGLSHPVALTFTKENSLLVLDTASHSIKVYKVDWGSEQ ID NO: 44-Human Pancreatic Alpha Amylase AminoAcid Sequence (GenBank Accession No.: NP_000690.1)MKFFLLLFTIGFCWAQYSPNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIYNPFRPWWERYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFNPGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLTGLLDLALEKDYVRSKIAEYMNHLIDIGVAGFRLDASKHMWPGDIKAILDKLHNLNSNWFPAGSKPFIYQEVIDLGGEPIKSSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFVPSDRALVFVDNHDNQRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRQFQNGNDVNDWVGPPNNNGVIKEVTINPDTTCGNDWVCEHRWRQIRNMVIFRNVVDGQPFTNWYDNGSNQVAFGRGNRGFIVFNNDDWSFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSISNSAED PFIAIHAESKLSEQ ID NO: 45-Human Salivary Alpha Amylase AminoAcid Sequence (GenBank Accession No.: AAI44453.1)MKLFWLLFTIGFCWAQYSSNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWERYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFNPGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLSGLLDLALGKDYVRSKIAEYMNHLIDIGVAGFRIDASKHMWPGDIKAILDKLHNLNSNWFPEGSKPFIYQEVIDLGGEPIKSSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDNQRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRYFENGKDVNDWVGPPNDNGVTKEVTINPDTTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQVAFGRGNRGFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSISNSAED PFIAIHAESKLSEQ ID NO: 46-heavy chain variable domain CDR2 ofcertain antibodies of the disclosure, inaccordance with CDRs as defined by Kabat YISSGSSTIYYADSVKGSEQ ID NO: 47 -light chain variable domain CDR1 ofcertain antibodies of the disclosure, inaccordance with CDRs as defined by Kabat RASKSVSTSSYSYLASEQ ID NO: 48 -light chain variable domain CDR2 ofcertain antibodies of the disclosure, inaccordance with CDRs as defined by Kabat YASYLQS

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject disclosure have beendiscussed, the above specification is illustrative and not restrictive.Many variations of the disclosure will become apparent to those skilledin the art upon review of this specification and the claims below. Thefull scope of the disclosure should be determined by reference to theclaims, along with their full scope of equivalents, and thespecification, along with such variations.

1.-10. (canceled)
 11. A method of treating Lafora Disease in a subjectin need thereof, comprising administering a composition comprising: (i)an acid alpha-glucosidase (GAA) polypeptide (e.g., a GAA polypeptidecomprising or consisting of mature GAA) and (ii) an internalizing moietythat promotes delivery into cells, wherein the internalizing moiety cantransit cellular membranes via an equilibrative nucleoside transporter 2(ENT2) transporter, and wherein the GAA polypeptide is interconnectedwith the internalizing moiety.
 12. A method of decreasing glycogenaccumulation in cytoplasm of cells of a subject having Lafora Disease,comprising administering to the subject a composition comprising (i) anacid alpha-glucosidase (GAA) polypeptide (e.g., a GAA polypeptidecomprising or consisting of mature GAA) and (ii) an internalizing moietythat promotes transport into cytoplasm of cells via an equilibrativenucleoside transporter 2 (ENT2) transporter, wherein the administeredcomposition contacts neuronal cells, and wherein the GAA polypeptide isinterconnected with the internalizing moiety.
 13. The method of claim11, wherein the subject has a mutation in the EPM2A gene.
 14. The methodof claim 11, wherein the subject has a mutation in the EPM2B gene.15.-17. (canceled)
 18. The method of claim 11, wherein the chimericpolypeptide does not comprise the portion of GAA polypeptide set forthin residues 1-57 of SEQ ID NO: 1 or
 2. 19. The method of claim 11,wherein the chimeric polypeptide lacks at least a portion of the GAAfull linker region, wherein the full linker region corresponds to theamino acids 57-78 of SEQ ID NOs: 1 or
 2. 20. (canceled)
 21. The methodof claim 11, wherein neither the GAA polypeptide nor the chimericpolypeptide comprise a contiguous amino acid sequence corresponding tothe amino acids 1-60 of SEQ ID NO: 1 or
 2. 22. The method of claim 11,wherein the chimeric polypeptide or GAA polypeptide comprises the aminoacid sequence of SEQ ID NO:
 21. 23. The method of claim 11, whereinneither the GAA polypeptide nor the chimeric polypeptide comprise acontiguous amino acid sequence corresponding to the amino acids 1-66 ofSEQ ID NO: 1 or
 2. 24. The method of claim 11, wherein the chimericpolypeptide or GAA polypeptide comprises the amino acid sequence of SEQID NO:
 22. 25. The method of claim 11, wherein neither the GAApolypeptide nor the chimeric polypeptide comprise a contiguous aminoacid sequence corresponding to the amino acids 1-69 of SEQ ID NO: 1 or2.
 26. The method of claim 11, wherein the chimeric polypeptide or GAApolypeptide comprises the sequence of SEQ ID NO:
 23. 27.-185. (canceled)186. A composition comprising (i) an acid alpha-glucosidase (GAA)polypeptide (e.g., a GAA polypeptide comprising mature GAA) and (ii) aninternalizing moiety, wherein the internalizing moiety promotes deliveryof the composition into cells via ENT2, and wherein at least 90% of theGAA polypeptide is interconnected with the internalizing moiety.187.-219. (canceled)
 220. The composition of claim 186, wherein thechimeric polypeptide composition does not comprise the portion of GAApolypeptide set forth in residues 1-57 of SEQ ID NO: 1 or
 2. 221.(canceled)
 222. (canceled)
 223. The composition of claim 186, whereinneither the GAA polypeptide nor the composition comprise a contiguousamino acid sequence corresponding to the amino acids 1-60 of SEQ ID NO:1 or
 2. 224. The composition of claim 186, wherein the composition orGAA polypeptide comprises the amino acid sequence of SEQ ID NO:
 21. 225.The composition of claim 186, wherein neither the GAA polypeptide northe chimeric polypeptide composition comprise a contiguous amino acidsequence corresponding to the amino acids 1-66 of SEQ ID NO: 1 or 2.226. The composition of claim 186, wherein the chimeric polypeptidecomposition or GAA polypeptide comprises the amino acid sequence of SEQID NO:
 22. 227.-243. (canceled)
 244. The composition of claim 186,wherein the internalizing moiety comprises an antibody or antigenbinding fragment that can transit a cellular membrane via anequilibrative nucleoside transporter 2 (ENT2) transporter and/or bindsDNA with a KD of less than 100 nM. 245.-248. (canceled)
 249. Thecomposition of claim 244, wherein the antibody or antigen bindingfragment comprises a heavy chain variable domain comprising an aminoacid sequence at least 95% identical to SEQ ID NO: 9, or a humanizedvariant thereof; and/or wherein the antibody or antigen binding fragmentcomprises a light chain variable domain comprising an amino acidsequence at least 95% identical to SEQ ID NO: 10, or a humanized variantthereof. 250.-365. (canceled)